Compounds, composition, methods, targets for cancer therapy

ABSTRACT

This invention describes methods and pharmaceutical compositions for combinational cancer treatments that are capable of inducing JNK phosphorylation and induce programmed cell death. It also identified genes as target for anti-cancer drug development and enhancement of the chemotherapeutic drug effect for the treatment of cancer. This invention points to a novel method and principle for a new avenue of developing more efficient and low or non cytotoxic cancer treatment.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.12/667,687, filed on Jan. 4, 2010, which is a Continuation in Part ofPCT/US2008/069106 filed on Jul. 2, 2008 which claims priority from U.S.Provisional Application Nos. 60/929,535, filed Jul. 2, 2007 and61/156,507, filed Mar. 1, 2009, which are herein incorporated byreference in their entirety.

GOVERNMENT INTERESTS

The research carried out in the present application was supported inpart by NIH. The government may have certain rights in the invention ofthe present application.

SEQUENCE LISTING

-   Attached .txt file entitled: PCT/US2008/069106-SequenceListing-   The sequence Listing contains the following sequences:-   Nucleotide sequence of pUC19 and pcDNA3;-   Nucleotide sequences: Transcripts of TRPC6 (NM_(—)004621), SH3PXD2B    (NM_(—)001017995),-   MAGI3 (NM_(—)152900), TMEM182 (NM_(—)144632),    C6orf108(NM_(—)199184);-   Peptide sequences: TRPC6 (NP_(—)004612), SH3PXD2B (NP_(—)001017995),    MAGI-3-   (NP_(—)690864), TMEM182 (NP_(—)653233), C6orf108 (NP_(—)954653);-   Double strand RNA sequences: siRNA1, siRNA2, and siRNA3.

DESCRIPTION OF THE INVENTION

1. Technical Field of the Invention

This invention relates to the fields of oncology and chemotherapy.Specifically, the invention provides novel compounds, methods,pharmaceutical composition and targets for more efficient and less ornon cytotoxic treatments of cancer.

2. Background Art of the Invention

Up to date, chemotherapy and radiation therapy are still the mainstreamfor cancer treatment. These treatments were based on targetingproliferating cells rather than cancer cells only, which is also thecause fundamental basis of lethal side effects from these treatments.Targeted therapy, a new generation of cancer treatment, is aimed totarget cancer specific changes of molecules and signaling pathways toinduce cancer cell death, but limit such effects on normal cells.Enormous efforts have been made in finding the targets and the ways oftargeting the targets inside the cells as a treatment. However, up todate, the success rate of this new generation is limited. One majorchallenge comes from the complexity of cellular regulation mechanismsand overlapping pathways inside the cells.

Aberrant Nuclear factor-kappa B (NF-κB) activation has been associatedwith a variety of tumors and cancer cells for oncogenesis, regulation ofcell proliferation, inhibition of apoptosis, promoting angiogenesis,tumor invasion and metastasis as well as cancer cell resistance tochemotherapy and radiation therapy treatments (Kim H J, Hawke N, andBaldwin A S, NF-κB and IKK as therapeutic targets in cancer, Cell Deathad Differentiation, (2006) 13:738-47; Karin M, Nuclear factor-κB incancer development and progression, (2006) Natur441:431-6) InhibitionNF-κB activity could facilitate cancer cell death and sensitize cancercells to chemotherapy drugs and radiation therapy (Kim H J, Hawke N, andBaldwin A S, NF-κB and IKK as therapeutic targets in cancer, Cell Deathand Differentiation, (2006) 13:738-47; Karin M, Nuclear factor-kB incancer development and progression, (2006) Natur441:431-6; ChikashiNakanishi and Masakazu Toi, Nuclear Factor-κB Inhibitors As SensitizersTo Anticancer Drugs, NATURE REVIEWS CANCER (2005) 5:297-309).

There are two similar, but different IκB Kinases (IKK1 and IKK2) thatare up stream regulator of NF-κB activity. In addition, alternativeNF-κB activation pathways, such as protein kinase CK2 (CK2), also exist.In most cancer cells, NF-κB is constitutively activated. In addition tothe IKK classic pathway, these alternative NF-κB activation pathways mayalso contribute to the aberrant NF-κB activity in cancer cells (Ming Yu,Jason Yeh, and Carter Van Waes Protein Kinase CK2 MediatesInhibitor-Kappa B Kinase and Aberrant Nuclear Factor-κB Activation bySerum Factor(s) in Head and Neck Squamous Carcinoma Cells CancerResearch, 2006 Jul. 1; 66(13): 6722-6731. and other for NFκBactivation). Dozens of IKK inhibitors have been produced and are intrials for treating anti-inflammatory diseases. However, for treatingcancer with these IKK inhibitors, these efforts have yielded results farfrom spectacular (Chikashi Nakanishi and Masakazu Toi, Nuclear Factor-κBInhibitors As Sensitizers To Anticancer Drugs, NATURE REVIEWS CANCER(2005) 5:297-309).

More recent studies pointed to the balance between NF-κB activity andC-Jun-N-Terminal-kinase (JNK) activity, which regulates cell death orproliferation (reviews). In this theory, NF-κB and JNK cross talkthrough reactive oxygen species (ROS). Both JNK and NF-κB activity leadsto Cell proliferation. However, ROS induces prolonged JNK activationthat will induce programmed cell death. Conversely, activated NF-κBsuppresses ROS and, hence, suppress ROS induced prolonged JNKactivation. Therefore, inhibiting NF-κB while activating JNK wouldswitche the balance to programmed cell death. However, up to date, nosuch treatment method has been reported. Most importantly, although thisspecific theory has been proposed, none has prior succeeded indemonstrating this effect.

ROS are potentially harmful by-products of normal cellular metabolismthat directly affect cellular functions. ROS are also acts messenger andindispensable for signal transduction pathways that regulate cell growthand reduction-oxidation (redox) status. However, overproduction of thesehighly reactive oxygen metabolites can initiate lethal chain reactions,which involve oxidation and damage to structures that are crucial forcellular integrity and survival. In fact, many antitumor agents, such asvinblastine, cisplatin, mitomycin C, doxorubicin, camptothecin,inostamycin, neocarzinostatin and many others exhibit antitumor activityvia ROS-dependent activation of apoptotic cell death, suggestingpotential use of ROS as a fundamental antitumor principle. The“oxidation therapy” a unique anticancer strategy by inducing thegeneration of ROS directly to solid tumors as cytotoxic oxystress forcancer treatment has been developed. However no successful and practicalresults were obtained probably because of the lack of tumor selectiveROS delivery and hence resulting in subsequent induction of severe sideeffects (Fang, J., Nakamura, H., and Iyer, A. K Tumor-targeted inductionof oxystress for cancer therapy. J Drug Target, 15: 475-486, 2007).

One of the unique features of cancer cells is their dependency onaerobic glycolysis, the “Warburg effect” that most cancer cellspredominantly produce energy by glycolysis followed by lactic acidfermentation in the cytosol, rather than oxidation of pyruvate inmitochondria by most normal cells (Warburg 0., Science 123:309, 1956).Along with this Aerobic glycolysis is that cancer cells consume oxygenthrough trans-plasma membrane electron transport (tPMET) at cell surfacethat oxidizes the NADH⁺ that generated from the glycolysis processes incytosol and to generate ATP (Heart, P M, Curr Mol Med, 2006, 6:895). ThetPMET is mediated by NADH Oxidases (NOX) located on cell plasmamembrane. This process oxidizes intracellular NADH and recycles it tomaintain the intracellular NADH/NAD+ ratio to support glycolytic ATP. AsATP production contributes substantially to fulfilling the energyrequirements of rapidly dividing cells, such as cancer cells, and thattPMET is the major source for cancer cell energy production that isdifferent from normal cells, which perform energy metabolism and consumeoxygen in mitochondrial. Therefore, targeting tPMET could be a strategyfor cancer specific treatment. This concept was initially proposed byHerst P M, and Berridge M V based on the facts that the compounds thataffect tPMET also affect cancer cell survival (Herst P M, Berridge M V,Curr Mol Med 6:895, 2006). It was further hypothesed that blocking theelectron transport through interfering with membrane ubiquitourecycling, destabilizing the redox status of the cell membrane that maystimulate acid sphingo-myelinase activity, result in the conversion ofsphigomyelin to ceramide that will lead to formation ofceramide-enriched membrane islands, which lead to apoptosis (Dumitru, C.A. et al, 2006, Oncogene 25:5612-25). Based on this hypothesis, Berridgeet al proposed to make drugs specifically located to the plasma membranewithout entering the cell as a novel anticancer drug developmentstrategy. However, up to the date of filing this application, no suchdevelopment had been reported.

Hypoxia inducing factor (HIF) and pyruvate kinase 2(PK-M2) are known tobe responsible to the switch to aerobic glycolysis by cancer cells, buttargeting PK-M2 resulted intolerable side effects. High HIF expressionlevels and activities have been associated with all cancer cells thatmake cancer cells resistant to low oxygen levels. Furthermore, cancercells are actively undergoing catabolism, which result high demands forreducing sources that oxidize the NADH+ generated from the glycolysisprocess, which further makes cancer cells can survival in close to zerooxygen levels. Single inhibition of HIF seems not sufficient to killcancer cells. More effective inhibition of cancer cell specificrespiration is still lacking and has been sought hardly.

Apigenin is a naturally occurring plant flavone(4′,5,7,-trihydroxyflavone) abundantly present in common fruits andvegetables including apple, parsley, onions, oranges, tea, chamomile,wheat sprouts and some seasonings. Apigenin is a multi function signalconduction agent and has been shown to possess remarkableanti-inflammatory, antioxidant and anti-carcinogenic properties and iscurrently under active study. Studies on the biological effects ofapigenin at cellular and molecular levels have found that apigenininterferes with a wide range of critical molecules and signaling andregulatory processes in the cells, including depleting the HER2 proteinand suppressing the Her2/Her3-phosphatidylinositide 3-kinase/AKT pathway(Way, T D. and Lin, J. K Role of HER2/HERS co-receptor in breastcarcinogenesis. Future Oncol, 1: 841-849, 2005), inhibit HIF, PKC, CDK,VEGF NF-B, CK2, AKT, MAPK, AR and ER pathways, activate wild type p53,modulate the deregulated cell cycle checkpoint and induce apoptosis(Induction of caspase-dependent, p53-mediated apoptosis by apigenin inhuman neuroblastoma—Torkin et al. 4 (1): 1—. 2007; Apigenin InhibitsExpression of Vascular Endothelial Growth Factor and Angiogenesis inHuman Lung Cancer Cells: Implication of . 2007; Apigenin inhibits VEGFand HIF-1 expression via PI3K/AKT/p70S6K1 and HDM2/p53 pathways—Fang etal. 19 (3): 342—The FASEB. 2007; Balasubramanian, S, and Eckert, R. L.Keratinocyte proliferation, differentiation, and apoptosis—differentialmechanisms of regulation by curcumin, EGCG and apigenin. Toxicol ApplPharmacol, 224: 214-219, 2007; Birt, D. F., Walker, B., Tibbels, M. G.,and Bresnick, E. Anti-mutagenesis and anti-promotion by apigenin,robinetin and indole-3-carbinol. Carcinogenesis, 7: 959-963, 1986;Patel, D., Shukla, S., and Gupta, S. Apigenin and cancerchemoprevention: progress, potential and promise (review). Int J Oncol,30: 233-245, 2007; Sato, F., Matsukawa, Y, Matsumoto, K, Nishino, H.,and Sakai, T Apigenin induces morphological differentiation and G2-Marrest in rat neuronal cells. Biochem Biophys Res Commun 1994 Oct. 28;204: 578-584, 1994). In addition, apigenin has also been reported togenerate ROS, which disrupt mitochondrial membranes. Current researchtrials indicate that it may reduce DNA oxidative damage; inhibit thegrowth of human leukemia cells and induced these cells to differentiate;inhibit cancer cell signal transduction and induce apoptosis; act as ananti-inflammatory; and as an anti-spasmodic or spasmolytic. More than100 patent applications related to apigenin have been filed. Amongthose, apigenin was claimed to be used as a drug for treatinginflammatory and autoimmune diseases. In addition, apigenin was alsoclaimed for the use as a cancer chemoprevention drug and as adjunct usefor enhancing the effects of chemotherapy drugs for cancer treatment at10 μM concentration (US Patent Application 20060189680). However, as achemo sensitizer, the efficacy effect of apigenin is limited. To be acytotoxic drug for treating cancer, apigenin has to be combined withother treatments. Other isoforms of apigenin, other flavonoids,isoflavonoids including, naturally existed, modified or syntheticincluding phenoxodiol a synthetic isoflevene, have also been found withsimilar function of apigenin All of those need to be combined withchemotherapy drugs for cancer treatment.

SUMMARY OF THE INVENTION

A more efficient and cancer specific anticancer treatment can beachieved by combining inhibition of cancer cell surface respiration withinhibiting its hypoxia response.

The present invention provides pharmaceutical composition andcombinational composition and methods for the treatments of cancer andgenes as drug targets to enable the treatment of cancer in a mammal tosynergize cancer specific cell death with less or no cytotoxic sideeffects including:

A pharmaceutical composition and a method for treating cancer bytargeting the tPMET of cancer cells to block the tarns plasma membraneelectron transfer and/or uncoupling the oxidative phosphorylation acrossthe cell plasma membrane without affecting the same function at themitochondria membrane in combination inhibition of cellular responses tohypoxia to reach a synergistic therapeutic effect of inducing cancerspecific cell death for cancer treatment;

A compound and its required chemical structure for targeting tPMET forcancer treatment;

A use of WST-3 and any of the valid substitutes that are capable ofblocking the tPMET by uncoupling the oxidative phposphorylation on cellplasma membrane for the said combination treatment;

A pharmaceutical composition and a method for treating cancer bycombining WST-3 or its valid substitutes with apiginin or its validsubstitutes as an cancer specific and less toxic anticancer treatment;

A use of a reagent WST-1r comprising water soluble tetrozolium salts andintermediate electron acceptors as a drug to interfere tPMET for thesaid combination treatment;

A pharmaceutical composition of WST-1r and any of the valid substitutesof WST-1r for the said combination treatment that are capable ofconducting trans-plasma membrane electron transport and induces ROS. TheWST-1r and any of the valid substitutes of WST-1 is a mixture oftetrazolium salt and an electron coupling reagent (IEA), or at least oneof the tetrozolium salt or at least one of the IEA in optimizedconcentration. The compounds may be administered in a pharmaceuticallyacceptable carrier medium.

A pharmaceutical composition and a method for treating cancer bycombining WST-1r or its valid substitutes with apiginin or its validsubstitutes as an cancer specific and less toxic anticancer treatment;

Selected genes, molecules, and polynucleotide sequences and polypeptidesequences are provided as target for designing drugs for the treatmentof a cancer in a patient in need. These targets are the humantranscripts, and their corresponding protein/peptide molecules and/orthe genomic DNA sequences that are selected from the blast analysis ofthe DNA sequence of pUC19 DNA vector against human genome andtreanscripts, the DNA sequences of which mapped to the human transcriptsand/or genomic sequences in short pieces. The transcripts and theircorresponding coding molecules are targets for enhancing the efficacy ofthe treatments of cancer. Other sequences that, thus, mapped to humangenomic sequences may be used as targets as well as being used fortargeting these corresponding genes. The potential drugs that can bedesigned to targeting these targets include, but not limited to, siRNA,small molecule inhibitors, peptides inhibitors, anti-sense RNA,anti-sense Oligo, antibodies, antibody fragments, proteins, dominantnegative DNA vectors and Interferon (IFN). In a particular embodiment ofthis invention, these targets are, but not limited to, polynucleotidesequences of TRPC6 (SEQ ID NO: 2), MAGI-3(SEQ ID NO: 4), TMEM182 (SEQ IDNO: 5), SH3PXD2B (SEQ ID NO: 3), or c60rf108 (SEQ ID NO: 14), and thepolypeptide sequences of TRPC6 (SEQ ID NO: 6), MAGI-3(SEQ ID NO: 8),TMEM182 (SEQ ID NO: 9), SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO:15). The sequence to target human genomic sequence and or transcriptsare, but not limited to, puc19 DNA vector (SEQ ID NO: 1), pc DNA3 vector(SEQ ID NO: 13), siRNA2 (SEQ ID NO: 10-12). Synthetic siRNA that againstthese target genes (SEQ ID NO: 2-5 and SEQ ID NO: 14) were selected fordemonstrating the potential use of these genes as a target for thecombination treatment for cancer;

A method for treating a cancer in a patient in need thereof comprisingadministering to the patient, concurrently or sequentially, atherapeutically effective amount of (1) at least one of the transfectionof puc19 DNA vector or administering at least one of the substitutes ofpuc19 DNA transfection and (2) at least one IKK inhibitor and (3) anadditional third agent, WST-1r or at least one of the valid substitutesof WST-1r, in a pharmaceutically acceptable carrier medium. Wherein saidcombination enhances the induction of cancer cell death while otherwiseany of these agents separately are demonstrated not to be toxic.

The valid substitutes for Puc19 DNA transfection are selected from thegroup consisting of (1) type I IFN, (2) Synthetic small interfering RNAs(siRNA) the nucleotide sequence SEQ ID NO10-12 of which mapped to boththe DNA sequence of the pUC19 DNA vector and human transcripts andgenome DNA sequences, (3) the biological compounds selected from thegroup consisting of biological and non-biological organic or non-organiccompounds. The said method of screening compounds, wherein saidbiological chemicals are further selected from the group ofpolypeptides, proteins, peptides, antibodies, antibody fragments,nucleic acids, and polynucleotide the products of which interact andinterfere said selected targets of the polynucleotide sequences of TRPC6(SEQ ID NO: 2), MAGI-3(SEQ ID NO: 4), TMEM182 (SEQ ID NO: 5), SH3PXD2B(SEQ ID NO: 3), or c60rf108 (SEQ ID NO: 14), and the polypeptidesequences of TRPC6 (SEQ ID NO: 6), MAGI-3(SEQ ID NO: 8), TMEM182 (SEQ IDNO: 9), SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO: 15). SyntheticsiRNA that against these target genes (SEQ ID NO: 2 to 5 and SEQ ID NO:14) were selected for demonstrating the potential use of these genes asa target for the combination treatment for cancer;

A method of inducing programmed cell death of cancer cells in amalignant cell population, and treating a patient with cancer comprisingthe use of a combination therapy. The combination therapy of the presentinvention comprises administering an effective dose of WST-1r or anyvalid substitutes, that is capable of conducting trans-plasma membraneelectron transfer and induces ROS in a cell and apigenin, amulti-function inhibitor that inhibits HIF, CK2, NF-κB activity andother molecules and/or signaling pathways, or at least one of the IKKinhibitor. The said combination treatment enhances apigeninanti-neoplasm effect and synergizes the induced cancer cell death;

A method is provided for treating a cancer in a patient in need thereofby administering to the patient, concurrently or sequentially, atherapeutically effective amount of at least one GSK3 inhibitor andprotein kinase CK2 (CK2) inhibitor and addition of a third agent,WST-1r. In a particular embodiment of the invention, the preferred atleast one GSK3 inhibitor is LiCl and the preferred protein kinase CK2(CK2) inhibitor is Apigenin The compounds may be administered in apharmaceutically acceptable carrier medium. Wherein said combinationenhances the induction of cancer cell death otherwise any of theseagents separately are demonstrated not to be toxic;

A method is provided for treating cancer in a patient in need comprisingadministering, concurrently or sequentially, a therapeutically effectiveamount of a combination of a selective Puc19 DNA trasnfection or atleast one of any of the valid substitutes of puc19 transfection aslisted above in combination with at least one of a selected approvedchemotherapeutic agents. Wherein said Puc19 DNA trasnfection oradministering at least one of the valid substitutes of puc19transfection being capable of substantially enhancing anti-neoplasticeffects of said proved chemotherapeutic agents, substantially reducingtoxic side effects of said chemotherapeutic agents, or a combinationthereof, wherein said Puc19 DNA trasnfection or at least one of thevalid substitutes has a substantial effect on activity of saidchemotherapeutic agents;

A method is provided for synergistically inhibiting NF— BNF-KAPPABactivity in cancer cells and in a patient in need thereof byadministering to the cells or patient, concurrently or sequentially, atherapeutically effective amount of at least one Dominant negativekinase dead IKK1 DNA vector (IKK1-KA) and at least one Dominant negativekinase dead IKK2 DNA vector (IKK2-KA). The at least one Dominantnegative kinase dead IKK1-KA or IKK2-KA may be substituted by IKKinhibitors selected from the group consisting of (IKK inhibitor list).The compounds may be administered in a pharmaceutically acceptablecarrier medium. This combinational inhibition effect may be furtherenhanced by adding a third agent, WST-1r or the valid substitutes ofWST-1r, for further induction of cancer cell death;

A method of inducing cancer cell death, and treating a patientcomprising the use of a combination therapy. The combination therapy ofthe present invention comprises administering an effective dose of atleast a compound that inhibits NF-κB activity and at least one compoundthat inhibits STAT3 in a preferred embodiment, the compound thatinhibits NF-κB activity is apigenin or an IKK inhibitor or a CK2inhibitor and the compound that inhibits STAT is stattic. The compoundsmay be administered in a pharmaceutically acceptable carrier.

A use of the combination therapy to treat cancers comprisingadministering IKK inhibitor or apigenin and a STAT3 inhibitor, stattic.In one preferred embodiment, the cancers are selected from the groupconsisting of a subtype of head and neck squamous carcinoma.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Chemical Structure of DNP and WST-3

FIG. 2. Chart of endogenous NF-κB down stream gene expression levels.

FIG. 3. Chart of cell survival from IKK-KA transfection in combinationwith or without WST-1 treatment.

FIG. 4. Chart of WST-1 promotes HT1080 Human Sarcoma cell death bytriple combination Treatment

FIG. 5. Chart of combination of LiCl and Apigenin and WST-1r treatment.

FIG. 6. Chart of pUC19 DNA transfection synergize chemotherapeutic drugeffect

FIG. 7. Chart of Time Course of ROS generation after combinationtreatment of WST-1r, CCK8 with apigenin and IKK inhibitor III

FIG. 8. Chart of combination treatment with apigenin and WST-1rsynergizes induced cancer cell death.

FIG. 9. Chart for Differential Responses to WST-1r and ApigeninCombination Treatment from Human Non-Cancer Cells and Human Head andNeck Cancer Cells.

FIG. 10. Chart of Time course and Dose Response of WST-1r andDose-Response of apigenin involved in the combination treatment ofWST-1r with apigenin.

FIG. 11. Chart showing Effect of Combination treatment with IKKInhibitor and WST-1r on melanoma cell lines

FIG. 12. Chart of effects of treatment order of WST-1r and BMS345541 oninduced cell death

FIG. 13. Chart showing WST-1r and Apigenin combination treatment inducedJNK Phosphrylation

FIG. 14. Chart of Effects of CCK8 and XTT as substitute of WST-1r incombination treatment with apigenin for inducing cancer cell death

FIG. 15. Chart of Combination Treatment of Apigenin with WSTderivatives, mPMS or Combination of WST and mPMS

FIG. 16. Chart of Dose-Response of Apigenin and mPMS CombinationTreatment

FIG. 17. Chart of Differential cellular responses to mPMS treatment

FIG. 18. Chart of Effect of Combination WST-3 with Apigenin On CellDeath

FIG. 19. Chart of Effect of WST-3, and WST-3+mPMS in Combination withApigenin On Inducing Cancer Cell Death

FIG. 20. Chart of siRNA substitution of pUC19 for enhancing WST-1r-IKKinhibitor combination Treatment Effect

FIG. 21. Chart for Effect of Type I INF Substitute pUC19 for combinationCancer treatment

FIG. 22. Chart of enhancement of Taxel Efficacy Effects by Combinationof Puc 19 DNA sequence derived siRNA with Taxel

FIG. 23. Image of Induction od ROS Generation by WST-1r and thecombination treatment.

FIG. 24. Chart of Dose Response of ROS generation after combinationtreatment of WST-1r, CCK8 with apigenin and IKK inhibitor III

DETAILED DESCRIPTION OF THE INVENTION I. General Description

The efficacy of anticancer therapy can be enhanced by combination ofproper selected compounds, biological molecules and drugs that block thetPMET and cell surface respiration in combination with inhibitingcellular hypoxia responses to induce synergistic and cancer cellspecific cell death. Several combinational pharmaceutical compositionsand methods for anticancer treatment are described. The development ofthese pharmaceutical compositions were based on the followingdiscoveries:

The discovery the use of WST-3, WST-1r and their valid substitutes asdrugs for combination therapies by combining with apigenin, IKKinhibitors or Puc 19 DNA or any of its valid substitutes that inducedsynergetic and cancer specific cell death,

The discovery the structure and function features of WST-3 representinga class chemicals of a cell surface oxidative-phosphorylation uncpuplerand the corresponding principle to design a chemical compound fortargeting tPMET and blocking cell surface oxidative-phosphorylaition,tPMET and cell respiration for the said combination anticancertreatment.

(3) The use of puc19 DNA sequences and the corresponding siRNAs as anticancer drugs as well as the discovery of the corresponding genes astarget for developing anticancer therapy.

(4) The discovery of the method and combinational composition of WST-3and apigenin or their valid substitutes as anticancer treatment.

(5) The discovery of the method and combinational composition of WST-1rand apigenin or their valid substitutes as anticancer treatment.

The first and the second discoveries led to the identification ofclasses of chemicals and corresponding pharmaceutical compositions ofusing these chemical compounds as drugs for the combination treatmentfor cancer. The third finding further led to the discovery of severalgenes as targets for developing anticancer drugs. These are rarelystudied genes and some of them are still in hypothetical gene status.Together, these findings led to establishing several combinationaltreatment methods for cancer therapy.

In one embodiment, the pharmaceutical composition of WST-3 was describedas a cell surface oxidative-phosphorylation uncouple for the use asdrugs for combinational treatment of cancer.

In one embodiment, the pharmaceutical composition of WST-1r wasdescribed an agent that interferes tPMET for the use as drugs forcombinational treatment of cancer.

Yet in another embodiment, the classes of chemical compounds of WST-3and their special chemical structures for designing drugs to directtarget the tPMET and as an uncoupler to block the cell surface energymetabolism and cell surface respiration are descried for the use of thecombination treatment for cancer.

Yet in another embodiment, the classes of chemical compounds and thecombination of these compounds that can form the formula of WST-1r andthe valid substitutes of WST-1r are descried for the use of thecombination treatment for cancer.

In one of the embodiments, Puc19 DNA vector was found to have biologicaleffect on mammalian and human cancer cells and was used as a drug forcombination treatment with WST-1r reagents and with or without IKKinhibitors.

In another embodiment, Puc19 DNA vector was used as a drug incombination with chemotherapeutic drug for enhancing the therapeuticeffect of these chemotherapeutic drugs for the treatment of cancer.

According to the above embodiments, small interfering RNAs (SEQ-IDNo:10-12), the sequence of which were derived from the nucleotidesequence of Puc19 DNA vector, were described for the use of combinationtreatments for cancer.

Yet also according to the above embodiments, human genes (TRPC6 (SEQ IDNO: 2), MAGI-3(SEQ ID NO: 4), TMEM182 (SEQ ID NO: 5), SH3PXD2B (SEQ IDNO: 3), or c60rf108 (SEQ ID NO: 14), and the polypeptide sequences ofTRPC6 (SEQ ID NO: 6), MAGI-3(SEQ ID NO: 8), TMEM182 (SEQ ID NO: 9),SH3PXD2B (SEQ ID NO: 7), or c60rf108 (SEQ ID NO: 15)) that were selectedbased on Puc 19 DNA sequence analysis and the biological function of thecorresponding siRNAs to be used as target for drug development for thetreatment of cancer are described.

Yet another embodiment, wherein said the valid substitutes of Puc 19 DNAthat were selected from biological and non-biological compounds andtheir effects in combination with WST-1r or the valid substitutes ofWST-1r and with or without IKK inhibitor is described. Wherein said thebiological compound for the valid substitutes of Puc19 DNA includedifferent members of Interferon and all the siRNAs mentioned above.

Yet another embodiment, wherein said the valid substitutes of Puc19 DNAthat were selected from biological and non-biological compounds andtheir effects in combination with chemotherapeutic drugs is described.Wherein said the biological compound for the valid substitutes of Puc19DNA include different members of Interferon and all the siRNAs mentionedabove.

In another embodiment, a medical use of combination treatment for cancercomprising apigenin, the flavonoids or at least one IKK inhibitor andWST-1r is described.

Yet in another embodiment, a medcal use of combination treatment forcancer comprising at least one Protein kinase II (C1(2) inhibitor,apigenin, at least one GSK3 inhibitor, Lithium chloride, and WST-1r forenhancing treatment effect is described.

II. Definitions

The term “pUC19 DNA” is a DNA cloning vector (SEQ ID #1) that amplifiesin prokaryotic cells. DNA sequence of this vector was originallysubmitted to NCBI gene bank by J. Messing, Waksman Institute, NJ on3-MAR-1986 and revised by F. Pfeiffer on 16-DEC-1986. In the presentdescription, pUC19 has been used as a drug for anticancer therapy bytransfected into human cancer cells.

The data suggests that the DNA sequence that composes this DNA vectorhas biological effects in cultured human cancer cells that lead tosynergistic cell death when combined with other treatments to thesecells as described in this description. Blast analysis of the DNAsequence of pUC19 against human genome and transcripts for short matchesshowed multiple short sequences aligned to varies locations of flankingsequences of human genome and transcripts (Blast result is attached tothis application). In the present description, pUC19 represents thecombination of short DNA sequences, usually 15-100 bases that mapped tohuman transcripts and/or flanking regions of genes of human genome DNAsequences. Accordingly, the corresponding gene products are the targetsof the pUC19. The polynucleotide sequences and amino acid sequences thatinclude but not limited to siRNA, miRNA, shRNA, peptide that aredirectly derived from the pUC19 DNA sequence as well as derived from thecorresponding genes and small molecules and antibodies that can interactand/or inhibit the function and activity of these correspondingmolecules as direct gene products of their DNA sequences, the DNAsequences of their corresponding gene contain these short matched DNAsequences from the DNA sequence of pUC19. The matched DNA sequencesdon't have to be exact matches. The matched DNA sequences can varyslightly, 10%, 20%, and even up to 30-40%.

The term “pcDNA3m DNA” is a mammalian expression vector version 3.1 withmodifications [SEQ ID #13]. DNA sequence of this vector was originallyderived from the pUC19 with further modifications and obtained fromInvitrogen, which has discontinued the production and selling of thisvector. pcDNA3 has been transfected into human cancer cells by chemicalor liposome based DNA transfection reagents. Similar to pUC19, the DNAsequence that composes this DNA vector have biological effects incultured human cancer cells that lead to synergistic cell death whencombined with other treatments to these cells as described. In thepresent description, pcDNA3 represents the short DNA sequences, usually15-100 bases that mapped to human transcripts and/or human genome DNAsequences, and their corresponding gene products that include but notlimited to siRNA, miRNA, shRNA, peptide that are directly derived fromthe DNA sequence of this vector and small molecules that can interactand/or inhibit the function and activity of these correspondingmolecules as direct gene products of their gene sequences, the DNAsequences of their corresponding gene contain these short matched DNAsequences from the DNA sequence of pcDNA3. The matched DNA sequencesdon't have to be exact matches. The matched DNA sequences can vary up to30-40% changes.

The term “siRNA1” [SEQ ID #10] is a siRNA designed based on and derivedfrom the DNA sequence of pUC19 [SEQ ID #1]. This siRNA sequence matchesto the human transcript of Homo sapiens transient receptor potentialcation channel, subfamily C, member 6(TRPC6, GeneID: 7225), mRNA(gi1199232561NM_(—)004621.3) synonyms: TRP6, FSGS2, FLJ11098. In thepresent descripion, siRNA1 was used as a drug for targeting TRPC6 forthe treatment of cancer. As in general the siRNA sequence can varyslightly, 10%, 20% and even 30-40% of the exact sequence of thetranscript.

The term “siRNA3” [SEQ ID #12]. is a siRNA designed based on and derivedfrom the pUC19DNA sequence [SEQ ID #1]. This siRNA sequece matches tothe human transcript of Homo sapiens membrane associated guanylatekinase, WW and PDZ domain containing 3 (MAGI3, GeneID: 260425),transcript variant 2, mRNA (NM_(—)152900.1) synonyms: MAGI-3, MGC163281and the Homo sapiens transmembrane protein 182 (TMEM182, GeneID:130827), mRNA (NM_(—)144632.2). In the present description, siRNA3 wasused as drug for targeting MAGI3 and/or TMEM182 for the treatment ofcancer. As in general the siRNA sequence can vary slightly, 10%, 20% andeven 30-40% of the exact sequence of the transcript.

The term “siRNA2” [SEQ ID #11] is a siRNA designed based on and derivedfrom the DNA sequence of pUC19 [SEQ ID#1]. The ⅔ of this siRNA sequecematches to the human transcript of Homo sapiens SH3 and PX domains 2B(SH3PXD2B), mRNA. (SH3PXD2B, GeneID: 285590), mRNA (NM_(—)001017995)synonyms: HOFI; F1120831; KIAAl295. In addition, this sequence alsomapped to more than 45 sites within flankin sequences of human genome.In the present description, siRNA2 was used for targeting SH3PXD2B andall the other potential DNA sequences in the human genome for thetreatment of cancer. As in general the siRNA sequence can vary 30-40% ofthe exact sequence of the transcript.

The term “TRPC6” [Nucleotide SEQ ID #2, Peptide SEQ ID #6] representshuman transcript of Homo sapiens transient receptor potential cationchannel, subfamily C, member 6(TRPC6, GeneID: 7225), mRNA(NM_(—)004621.3) synonyms: TRPC6, FSGS2, FLJ11098. In the presentdescription, TRPC6 is a target for developing anticancer treatment.TRPC6 can be targeted by any means that alter its expression levels andactivities at functioning levels including but not limited to polynucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo,and dominant negative DNA vectors, peptide and amino acid sequences,such as peptide, and antibodies, and small molecule inhibitors. TheTRPC6 has been previous reported as a potential target for cancertreatment, but no report regarding the use of TRPC6 as a target for acombinational cancer treatment with IKK inhibitors, WST1r orchemotherapy drugs to reach the synergistic effect of promoting cancercell death. The siRNA1 sequence described above is the preferredsequence, but this does not limit from other siRNA sequences and othermeans as described in this paragraph. As in general the siRNA sequencecan vary slightly, 10%, 20% and even 30-40% from the exact sequence ofthe transcript.

The term “MAGI3” [Nucleotide SEQ ID #4, Peptide SEQ ID #8] representshuman transcript of Homo sapiens membrane associated guanylate kinase,WW and PDZ domain containing 3 (MAGI3, GeneID: 260425), transcriptvariant 2, mRNA (NM_(—)152900.1). Synonyms: MAGI-3, MGC163281. MAGI-3 islocalized with ZO-1 and cingulin at tight junctions in epithelial cells,whereas MAGI-3 was found in E-cadherin-based cell-cell contacts and infocal adhesion sites in primary cultured astrocytes (Adamsky K, ArnoldK, Sabanay H, Peles E., Junctional protein MAGIKK interacts withreceptor tyrosine phosphatase beta (RPTP beta) andtyrosine-phosphorylated proteins. (J Cell Sci. 2003, 116(Pt 7):1279-89).MAGI-3 interacts directly with LPA(2) and regulates the ability ofLPA(2) to activate Erk and RhoA MAGIKK regulates LPA-induced activationof Erk and RhoA (Zhang H, Wang D, Sun H, Hall R A, Yun C C, Cell Signal.2007 February; 19(2):261-8. Epub 2006 Aug 9). The function of MAGI3 hasbeen previous linked to cancer, but no report regarding the use of MAGI3as a target for a combinational cancer treatment with WST-1r, IKKinhibitors or chemotherapy drugs to reach the synergistic inhibition ofcancer cell growth and to promote cancer cell death. In the presentdescription, MAGI3 is a target for developing anticancer treatment.MAGI-3 can be targeted by any means that alter its expression levels andactivities at functioning levels including but not limited to polynucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo,and dominant negative DNA vectors, peptide and amino acid sequences,such as peptide, and antibodies, and small molecule inhibitors. ThesiRNA3 sequence described above is the preferred sequence, but this doesnot limit from other siRNA sequences and other means as described inthis paragraph. As in general the siRNA sequence can vary slightly, 10%,20% and even 30-40% from the exact sequence of the transcript.

The term “TMEM182” [Nucleotide SEQ ID #5, Peptide SEQ ID #9] representsHomo sapiens trans-membrane protein 182 (TMEM182, GeneID: 130827), mRNA(NM_(—)144632.2). In the present description, SH3PXD2B is a target fordeveloping anticancer treatment. TMEM182 can be targeted by any meansthat alter its expression levels and activities at functioning levelsincluding but not limited to poly nucleotides, such as siRNA, shRNA,anti-sense RNA, anti-sense DNA oligo, and dominant negative DNA vectors,peptide and amino acid sequences, such as peptide, and antibodies, andsmall molecule inhibitors. The siRNA3 sequence described above is thepreferred sequence, but this does not limit from other siRNA sequencesand other means as described in this paragraph. As in general the siRNAsequence can vary 10%, 20% and even 30-40% from the exact sequence ofthe transcript. TMEM182 has not been previously studied and not beenlinked to cancer.

The term “SH3PXD2B” [Nucleotide SEQ ID #3, Peptide SEQ ID #7] representsSH3 and PX domains 2B adaptor protein HOFI (GeneID: 285590) thatcontains SH3 and PX domains. SH3 domains, Src homology 3 domains, bindto prolinerich ligands with moderate affinity and selectivity,preferentially to PxxP motifs; they play a role in the regulation ofenzymes by intramolecular interactions, changing the subcellularlocalization of PX; PhoX homologous domain, present in p47phox andp40phox. Eukaryotic domain of unknown function presents in phoxproteins, PLD isoforms, and a PI3K isoform. SHPXD2B has not beenpreviously studied and not been linked to cancer. In the presentdescription, SH3PXD2B is a target for developing anticancer treatment.SH3PXD2B can be targeted by any means that alter its expression levelsand activities at functioning levels including but not limited to polynucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo,and dominant negative DNA vectors, peptide and amino acid sequences,such as peptide, and antibodies, and small molecule inhibitors. ThesiRNA2 sequence described above is the preferred sequence, but this doesnot limit from other siRNA sequences and other means as described inthis paragraph. As in general the siRNA sequence can vary 10%, 20% andeven 30-40% from the exact sequence of the transcript.

The term “C6orf108” [Nucleotide SEQ ID #14, Peptide SEQ ID #15]represents human C6orf108 chromosome 6 open reading frame 108 [Homosapiens] GeneID: 10591. Official Symbol C6orf108. This gene wasidentified on the basis of its stimulation by c-Myc protein. The exactfunction of this gene is not known but studies in rat suggest a role incellular proliferation and c-Myc-mediated transformation. In the presentdescripion, C6orf108 is a target for developing anticancer treatment.C6orf108 can be targeted by any means that alter its expression levelsand activities at functioning levels including but not limited to polynucleotides, such as siRNA, shRNA, anti-sense RNA, anti-sense DNA oligo,and dominant negative DNA vectors, peptide and amino acid sequences,such as peptide, and antibodies, and small molecule inhibitors.

The term “Interferon” (IFN) is a group of cytokines produced byleucocytes and fibroblasts. The IFN that are described herein includesall type I and type II IFNs and all the subtypes of IFN including, butnot limited to IFN A, IFN B, IFN C, IFN D, IFN F, IFN G, IFN H, IFN I,IFN J, IFN K, IFN 4b, IFN WA, IFN IFN and IL-6.

The term “WST-1c” representing a water soluble tetrazolium salt WST-1{4-[3-(4-Iodopheny0-2-(4-nitropheny0-2H-5-tetrazolio]-1,3-benzenedilsulfonate}was first described by ishiyama et al in 1996 (Ishiyam M, et al BiolPharm Bull 1996, 19:1515-20).

The term “WST-1r” represents a reagent mixture comprising WST-1c andmPMS at optimized concentration and the ratio between WST-1c and mPMSfor the combination treatment. The optimized concentration and molarration of the two components may not be the same as that of thecommercial “cell proliferation kit”

The term “IEA” is the symbol of “Intermediate Electron Acceptor”.

The term “mPMS” (1-methoxy-5-methyl-phenazinium methyl sulfate) is achemical compound acts as an “electron coupling agent/IEA” when combinedwith tetrazolium salts.

The term “Q1” (coenzyme Q1,2,3-Dimethoxy-5-methyl-6-(3-methyl-2-butenyl)-1,4-benzoquinone) is achemical compound act as an IEA.

The term “WST” represents the collection of a class of compounds ofwater soluble tetrazolium salts including, but not limited to WST-3,WST-4, WST-5, WST-8, WST-9, WST-10, WST-11, XTT, and MSN. Thesecompounds are also impermeable to cell plasma membrane.

The term “XTT” represents a water soluble tetrazolium salt in thesimilar class of WST-1 as well as a reagent that composed of XTT andmPMS or coenzyme Q1.

The term “CCK8” represents a cell counting kte, which is composed ofWST-8 and mPMS.

The term “valid substitutes of WST-1r” represents any compounds that cansubstitute the function of WST-1r, WST-1c, or the electron couplingreagent mPMS or any of the remaining components either act alone or inany type of combination among these substitutes or any type ofcombination with any of the component of the water soluble tetrazoliumsalt and IEA that comprising WST-1r to function as the WST-1r asdescribed in this specification to reproduce the synergistic inductionof cancer cell death. The term “valid substitutes of WST-1r” includes,but not limited to all the up to date available tetrazolium salt basedWSTs that include, but not limited to, WST-1, WST-3, WST-4, WST-5,WST-9, WST-10 AND WST-11, MTS and XTT, and an IEA, including mPMS andcoenzyme Q1 and the combination of these tetrazolium salts with IEAcomprising WST-1+mPMS, WST-3+mPMS, WST-4+mPMS, WST-5+mPMS, WST-9+mPMS,WST-10+mPMS, WST-11+mPMS, XTT+mPMS, MTS+mPMS, WST-3+Q1, WST-4+Q1,WST-5+Q1, WST-9+Q1, WST-10+Q1, WST-11+Q1, XTT+Q1 MTS+Q1.

The term “IKK inhibitor” refers to an agent capable of inhibiting theactivity of Inhibitor kappaB kinase (IKK) and thereby inhibiting thekinase activity of IKK and its function of activating NF-kB. Therefore,inhibits NF-κB activity. An IKK inhibitor may be a competitive,noncompetitive, or irreversible IKK inhibitor. “A competitive IKKinhibitor” is a compound or a peptide that reversibly inhibits IKKenzyme activity at the catalytic site; “a noncompetitive IKK Inhibitor”is a compound that reversibly inhibits IKK enzyme activity at anon-catalytic site; and “an irreversible IKK inhibitor” is a compoundthat irreversibly destroys IKK enzyme activity by forming a covalentbond with the enzyme. The term “IKK inhibitors” include, withoutlimitation, i) compounds previously established to exhibit IKKinhibitory properties including, but not limited to: SPC839 (SignalPharmaceutical Inc.), Anilino-Pyrimidine Derivative (SignalPharmaceutical Inc.), PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute, IKKinhibitor III), SC-514*(Smithkilne Beecham Corp.),Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.),Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidinederivative (Bayer), Pyridooxazinone derivative (Bayer),Indolecarboxamide derivative (Aventis Pharma), Benzoimidazolecarboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinolinederivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehydesemicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), IKB Kinase Inhibitor Peptide (CalBiochem), IKK-2 InhibitorIV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide(CalBiochem), IKKInhibitor II, Wedelolactone (CalBiochem), IKK Inhibitor VII(CalBiochem), IKK-2 Inhibitor VN-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide IMD-0354(CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide (CalBiochem), IKK-2 InhibitorVIII ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxyphenyl)-4-(4-piperidinyl)-3pyridinecarbonitrile(CalBiochem). ii) In a certain embodiment, the group of IKK inhibitorsmay additionally include compounds discovered to have IKK inhibitoryactivity, in accordance with the present specification, and previouslyidentified to have anti-tumor activity, including, but not limited toPS1145(Millennium Pharmaceutical Inc.), BMS-345541*(Bristol-Myers SquibbPharmaceutical Research Institute).

The term “CK2 inhibitor” represents all protein kinase casein kinase2inhibitors. The preferred CK2 inhibitors is, but not limited to Apigenin

The term “Apigenin” CAS Registry Number: 520-36-5, Chemical AbstractsService Name:4H-1-benzopyran-4-one,5,7-dihydroxy-2-(4-hydroxy-phenyl)-(9CI). It isalso named as Apigenine; Chamomile; Apigenol; Spigenin; and Versulin andis a member of Flavones, a subclass of flavonoids. Apigenin is a multifunction signal transductor modulator that reduces DNA oxidative damage;inhibit the growth of human leukemia cells and induced these cells todifferentiate; inhibit cancer cell signal transduction and induceapoptosis; act as an anti-inflammatory; and as an anti-spasmodic orspasmolytic. Apigenin inhibits activity of NF— B, IKK-1 and IKK-2,protein kinase 2 (C1(2), mape kinase (MPK), hypoxia inducing factor 1(HIF), vescular epithelium growth factor (VEGF) and some other moleculesand regulatory pathways such as cell cycle and angiogenesis, induce p53activity, maintaining genomic stability by holding cell cycle formismatch repair or arrest cell cycle and induce apoptosis etc. Apigeninis know to have the effects of anti-UV radiation caused oxidation, andchemoprevention for cancer. The apigenin, herein, is also described as arepresentative of the subclasses of flavonoids, the flavones including,but not limited to: tricin, luteolin, tangeritin, 6-hydroxyflavone,Baicalein, Scutellarein, Wogonin, Diosmin, Flavoxate, Chrysin, theglycosided forms of these flavones, and other subclasses of theflavonoids with similar biological activities include, but not limitedto Isoflavones, Flavonols, Flavanones, 3-Hydroxyflavanones,Flavan-3-ols, Anthocyanidins, 3-deoxyanthocyanidin, Anthocyanins,Acetylated and glycosides, and Tannins, as well as isoflavonoids andneoflavonoids.

The term “Flavonoids” also called bioflavonoids also collectively knowas Vitamin P and citrin, are a class of plant secondary metabolites.Herein flavonoids represent all of the three ketone-containing compounds(flavonoid and flavonols) according to IUPAC nomenclatureclassifications: i) the flavonoids derived from 2-phenylchromen-4-one(2-phenyl-1,4-benzopyrone) structure; ii) isoflavonoids, derived from3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure; and iii)neoflavonoids, derived from 4-phenylcoumarine(4-phenyl-1,2-benzopyrone)structure; as well as the non-ketone polyhydroxy polyphenol compoundsincluding: flavanoids, flavan-3-ols and catechins Sample compoundsinclude, but not limited to Isoflavone:Biochanin A, Daidzein, Daidzin,Formononetin, Genistein, Coumestrol, Puerarin; flavan-3-ols: catechins(catechin, epicatechin (EG), epicatechin, gallate (EGC), andepigallocatechin gallate (EGCG)); flavonol: myricetin, quercetin, andKaempferol; Isoflavenes: phenoxodiol; Anthocyanins: Antirrhinin,Chrysanthenin, Malvin, Myrtillin, Oenin Primulin, Protocyanin,Tulipanin; 3-deoxyanthocyanidin: Apigeninidin, Columnidin,Diosmetinidin, Luteolinidin, Tricetinidin; Anthocyanidins: Aurantinidin,Cyanidin, Delphinidin, Europinidin, Luteolinidin, Malvidin,Pelargonidin, Peonidin, Petunidin, Rosinidin; 3-Hydroxyflavanones:Dihydrokaempferol, Dihydroquercetin; Flavanones: Eriodictyol,Hesperetin, Homoeriodictyol, Naringenin; Flavonols: Fisetin,Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin, Rhamnazin,Morin; and their glycoside forms.

The term “HIF” hypoxia inducible factor represents a family oftranscription factors that response to decrease of available oxygen orhypoxia in the cellular environment. Three family members have beenidentified. They are HIF-1 (a dimmer composed of HIF-1 and HIF-1), HIF-2(a dimmer composed of HIF-2 and HIF-2), HIF-3 (a dimmer composed ofHIF-3 and HIF-3).

The term “HIF inhibitors” are the biological and non-biologicalcompounds that inhibit HIFs and/or cellular responses to hypoxia,including, but not limited to: 2,2-dimethybenzopyran compounds,chetomin, 2-methoxyestradiol (2ME2), PX-478,17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968,camptothecins, NSC 644221,3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, anddecoy oligonucleotides against HIF-1 RX-0047.

The term “tNOX’ represents a tumor specific cell surface NADH oxidase.It is also called ECTO2.

The term “tNOX inhibitors” represents the compounds that are capable ofinhibiting the tNOX activity. The tNOX inhibits, herein, includes, butnot limited, catechins: catechin, epicatechin (EG), epicatechin gallate(EGC), and epigallocatechin gallate (EGCG); and a isoflavenes analoguederivative, the phenoxodiol.

The term “Oxidative Phosphorylation” is a process that coupling theoxidation of the protons with the synthesis of ATP, which transfer andstore the energy derived from glucose metabolism to the ATP as cellularenergy source.

The term “Uncoupler” means to uncouple the cellular oxidativephosphrylation process that blocks the ATP synthesis, the energymetabolism in the cell. The known unucouplers including, but not limitedto: dinitrophenol (DNP), Carbonyl cyanide m-chlorophenyl hydrazone(CCCP), Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP),Hindered pheniil (SF6847), Salicylanilide S-13, PCP, TTFB, andalpha-(phenylhydrazono)phenylacetonitrile derivatives.

The term “LiCl” is an inorganic salt, lithedium Chloride, and is used asan inhibitor of GSK3 LiCl, herein, represents the class of inhibitorsthat inhibit GSK3.

The term “IKK” represents Inhibitory kappaB Kinase, which phosphorylateI B that leads to NF-KAPPAB activation. Two IKK isoforms have beenidentified. They are IKK1 (IKK) and IKK2 (IKK). The term “NF-kappaB”Nuclear factor kappaB is a family of re1 proteins that act astranscription factors regulating gene expression. Normally NF-KAPPABproteins forms a dimmer which also complex with an inhibitory kappa B (IB) molecule stay in inactive form in the cytoplasm. Upon signalactivation, the I B is phosphorylated by IKK and dissociate from theNF-kappaB dimmer, which release the NF-KAPPAB to entering the nuclearfor activating transcription of a special set of genes that areregulated by NF-KAPPAB. The dissociated I B will be degraded byprotesomes. Activation of NF-kappaB favors cell proliferation andsurvival. NF-kappaB activity has been found to associate with andcontribute to carcinogenesis process, tumor progression and resistanceof cancer cells to chemo and radiation therapies.

The term “C-Jun N-terminal kinases” (JNKs), originally identified askinases that bind and phosphosphorylate c-Jun on Ser63 and Ser73 withinits transcriptional activation domain, are mitogen-activated proteinkinases which are responsive to stress stimuli, such as cytokines,ultraviolet irradiation, heat shock, and osmotic shock, and are involvedin T cell differentiation and apoptosis.

The term “Reactive Oxygen Species” (ROS) includes oxygen ions, freeradicals and peroxides both inorganic and organic. They are generallyvery small molecules and are highly reactive due to the presence ofunpaired valence shell electrons. ROSs form as a natural byproduct ofthe normal metabolism of oxygen and have important roles in cellsignaling. The effects of ROS on cell metabolism have been welldocumented in a variety of species. These include not only roles inprogrammed cell death and apoptosis, but also positive effects such asthe induction of host defence genes and mobilisation of ion transportsystems. This is implicating them more frequently with roles in redoxsignaling or oxidative signaling.

The term “Cancer Cells” represents the cells in culture that werederived from human cancer or tumors, which have malignant features, suchas lost of contact inhibition.

The term “Cancer” describes a diseased state in which a carcinogenicagent or agents causes the transformation of a normal cell into anabnormal cell, the invasion of adjacent tissues by these abnormal cells,and lymphatic or blood-borne spread of malignant cells to regional lymphnodes and to distant sites, i.e., metastasis.

The term “Effective dose” As used herein, the term “effective dose”means that amount of a drug or pharmaceutical agent that will elicit thebiological or medical response of a cell, tissue, system, animal orhuman that is being sought, for instance, by a researcher or clinician.

The term “therapeutically effective amount” means any amount which, ascompared to a corresponding subject who has not received such amount,results in improved treatment, healing, prevention, or amelioration of adisease, disorder, or side effect, or a decrease in the rate ofadvancement of a disease or disorder. The term also includes within itsscope amounts effective to enhance normal physiological function.

The term “Treatment of cancer” describes the drug or reagentsadministrated to the cells or to a mammal, the duration of thetreatment, the method used to administrate these drugs, or reagents andthe order and intervals of between these treatments.

The term “Synergistic effect/Synergize” refers to a combination of twoor more treatments, which is more effective to produce advantageousresults than the additive effects of these agents.

The term “Chemotherapy Drugs (Agent)” refers to any drugs that havecytrotoxic effect on cancer cells and are currently used as a drug fortreating cancer. The drugs that were tested in this specification arelisted as the following. Chemotherapy Drugs that we are mentioned inthis specification were not limit to this list.

The term “5-fluorouracil”,5-fluoro-2,4-(1H,3H) pyrimidinedione(5-FU), iscommercially available as fluorouracil.

The term “Cis-Platinum” cis-diamminedichloroplatinum, is commerciallyavailable as PLATINOL® as an injectable solution.

The term “Paclitaxel” is a potent anti-neoplastic drug; binds to theN-terminal region of β-tubulin and promotes the formation of highlystable microtubules that resist depolymerization, thus preventing normalcell division and arresting the cell cycle at the G₂/M phase.

The term “Doxorubicin”,(8S,10S)-10-[(3-amino-2,3,6-trideoxy-.alpha-L-lyxo-hexopyranosyl)oxy]-8-glycoloyl,7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12 naphthacenedionehydrochloride, is commercially available as an injectable form as RUBEX®or ADRIAMYCIN RDF®.

The term a “therapeutically effective amount” of a compound or apharmaceutical composition refers to an amount sufficient to modulatecancer cell proliferation in culture, tumor growth or metastasis in ananimal, especially a human, including without limitation decreasingtumor growth or size or preventing formation of tumor growth in ananimal. This term may also mean the effective amount(s) needed to causecancer cell death or selective cancer cell death while not causing sideeffects in normal cells.

The term “Pharmaceutically acceptable” indicates approval by aregulatory agency of the Federal or a state government or listed in theU.S. Pharmacopeia or other generally recognized pharmacopoeia for use inanimals, and more particularly in humans.

The term a “carrier” refers to, for example, a diluent, adjuvant,excipient, auxilliary agent or vehicle with which an active agent of thepresent specification is administered. Such pharmaceutical carriers canbe sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water or aqueoussaline solutions and aqueous dextrose and glycerol solutions arepreferably employed as carriers, particularly for injectable solutions.Suitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. It also include thetransfection reagents as used for deliver of DNA and/or RNA into cellseither in vitro or in vivo.

The term “Concurrently” means (1) simultaneously in time, or (2) atdifferent times during the course of a common treatment schedule.

The term “Sequentially” refers to the administration of one active agentused in the method followed by administration of another active agent.After administration of one active agent, the next active agent can beadministered substantially immediately after the first, or the nextactive agent can be administered after an effective time period afterthe first active agent; the effective time period is the amount of timegiven for realization of maximum benefit from the administration of thefirst active agent.

III Targets and Targeting the Therapeutic Targets for the Treatment ofCancer

This description provides nucleotide sequences for genes that implicateand/or can be utilized as therapeutic targets for the treatment ofcancer, and polypeptides encoded by such sequences and antibodies andcompounds reactive with such polypeptides in methods of treating acancer, and for agents effective in reducing the activity ofcancer-linked genes and thereby treating a cancerous condition whichwere not previously established for anti-tumor effect(s).

The disclosed nucleotide sequences are related to and derived from a DNAcloning vector, pUC19 (SEQ ID #1), which was discovered to synergize IKKinhibition, inhibit cancer cell growth proliferation and promote cancercell death when transfection of this vector to cancer cells was combinedwith or without IKK inhibitor treatment and followed by WST-1 r or anyof its valid substitutes treatment or in combination withchemotherapeutic drugs. This function of pUC19 has not been previouslyreported. Other potential DNA sequence may also include a pcDNA3 version3.1, (SEQ ID 13) and the attached blast result entitled: “NCBIBlast_pcDNA3 Nucleotide sequence (5448 letters)”.

Accordingly, the discovery that the anti-cancer effect of pUC19 vector(SEQ ID #1) was primarily resides in its DNA sequences that are mappedto transcripts and/or short sequences (from 15 bp up to 100 bp) thatflanking the genes in human genome. The human transcripts that pUC19 DNAsequences mapped to are, but not limited to, (1) Homo sapiens transientreceptor potential cation channel, subfamily C, member 6 (TRPC6, GeneID:7225, mRNA: NM_(—)004621.3, SEQ ID #2, #6), (2) Homo sapiens SH3 and PXdomains 2B (SH3PXD2B, GeneID: 285590, mRNA:NM_(—)001017995, SeQ ID #3,#7), (3) Homo sapiens membrane associated guanylate kinase, WW and PDZdomain containing 3 (MAGIKK, GeneID: 260425, transcript variant 2,mRNA:NM_(—)152900, SeQ ID #4, #8), (4) the Homo sapiens trans-membraneprotein 182 (TMEM182, GeneID: 130827, mRNA: NM_(—)144632, SeQ ID #5, #9)and (5) Homo sapiens chromosome 6 open reading frame 108 C6orf108,GeneID: 10591 SeQID #14, #15). The human genome sequences that pUC19 DNAsequences mapped to are listed in the attached file “NCBIBlast-pUC19-Human-Transcripts and genome(2686 letters)”, “NCBIBlast_siRNA2 Nucleotide sequence (24 letters)” and “NCBI Blast_pcDNA3Nucleotide sequence (5448 letters)”.

The polynucleotide disclosed herein incorporate various polynucleotidetranscripts (SEQ ID NO: 2, 3, 4, 5 and 14) and, thus, derived amino acidsequence (SEQ ID NO: 6, 7, 8, 9 and 15) from said transcripts areavailable as targets for treatment of cancer, especially anti-canceragents, including, with no limitation, peptide and proteins, such asantibodies specific against said polypeptides, peptide inhibitors, smallmolecule inhibitor, polynucleotides, such as siRNAs, shRNA, anti-senseRNA, anti-sense oligo and dominant negative DNA vectors. In a particularembodiment the wherein said double strand siRNAs are, but not limitedto, siRNA1 (SEQ ID #10), siRNA2 (SEQ ID #11), siRNA3 (SEQ ID #12).

The polynucleotides and polypeptides, as gene products, used in theprocesses may comprise a recombinant polynucleotide or polypeptide, anatural polynucleotide or polypeptide, or a synthetic polynucleotide orpolypeptide, or a chemically modified polynucleotide or polypeptide.

The nucleotides and polypeptides of the pUC19 vector, that are mapped tothe human genome, flanking genes in the human genome used in theprocesses of the present description may comprise a recombinantpolynucleotide or polypeptide, a natural polynucleotide or polypeptide,or a synthetic polynucleotide or polypeptide.

Fragments of such polynucleotide and polypeptides as are disclosedherein may also be useful in practicing the processes of the presentspecification. For example, a fragment, derivative or analog of thepolynucleotide (SEQ ID#2, 3, 4, 5 and 14) may be substituted by (i) anypart of these sequences and/or with mismatches for up to 40% of thetotal sequences been used for, (ii) fused into a DNA vector or any typeof carriers, (iii) nucleotide sequences with modified nucleotides.

Fragments of such polynucleotides and polypeptides as are disclosedherein may also be useful in practicing the processes of the presentspecification. For example, a fragment, derivative or analog of thepolypeptide (SEQ ID NO: 6, 7, 8, 9 and 15) may be (i) one in which oneor more of the amino acid residues are substituted with a conserved ornon-conserved amino acid residue (more preferably a conserved amino acidresidue) and such substituted amino acid residue may or may not be oneencoded by the genetic code, or (ii) one in which one or more of theamino acid residues includes a substitute group, or (iii) one in whichthe mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as a leader or secretorsequence or a sequence which is employed for purification of the maturepolypeptide (such as a histidine hexapeptide) or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Substituting these siRNAs (SEQ ID 10, 11, 12) as disclosed herein abovemay also be useful in practicing the processes of the presentspecification. Examples may include, but not limited to, (i) a siRNAthat mapped to another part of the sequence of the coding sequence ofthe gene, (ii) variations of the siRNA sequences that still capable totarget the same gene and reduce it expression level, (iii) any type ofmodifications of the siRNA either at the nucleotides or the whole siRNA,(iv) put the siRNA sequence into any type of carriers, such as a vectoror a chemical for the delivery of the sequence.

The nucleotide sequence of the complete mRNA and open reading frame ofthe transcripts and amino acid sequences, as discussed above, can befound in the NCBI GenBank database with the Gene ID or accession numberslisted above.

The pharmaceutical compositions and the medical use as described arebased, at least in part, on the discovery of inhibitory effect of pUC19vector in cancer cell growth and proliferation and inducing cancer celldeath when combined with IKK inhibitor WST-1r treatment as well as incombination with chemotherapeutic drugs to treat cancer cells. Thisinhibitory effect of pUC19 DNA transfection may be substituted by siRNA,compounds or small molecule inhibitor, peptide inhibitor, antibody,shRNA, anti-sense RNA, anti-sense oligo, and antibody and dominantnegative DNA vectors targeting the gene to alter its expression level,the corresponding transcripts and/or protein as described above in thissection and at least in partial by IFN.

Cancers that may be treated using the present discovery include, but arenot limited to: cancers of the prostate, colorectum, pancreas, cervix,stomach, endometrium, brain, liver, bladder, ovary, testis, head, neck,skin (including melanoma and basal carcinoma), mesothelial lining,esophagus, breast, lung (including small-cell lung carcinoma andnon-small-cell carcinoma), adrenal gland, thyroid, kidney, glioblastoma,mesothelioma, renal cell carcinoma, gastric carcinoma, choriocarcinoma,cutaneous basocellular carcinoma, and testicular seminoma, sarcoma ofmuscle, connective tissue or bone and leukemia.

IV. Pharmaceutical Compositions and Methods for Cancer Therapy

1. Inhibition of tPMET and Cell Surface Respiration in Combination withInhibition of HIF as a Strategy for Synergizing Cancer Cell Death as aCancer Treatment

A living cell relies on energy. Unlike normal cells that consume oxygenand generate ATP in mitochondrial, cancer cells consume oxygen on cellsurface through tPMET. This cellular geographic difference betweencancer cells and normal cells makes the PMET a unique site for cancerspecific targeting. In addition, cancer cells are resistant to hypoxiadue to increased levels and activities of hypoxia inducible factor(HIF). Therefore, blocking the PMET while inhibiting the HIF will inducesynergistic and cancer specific cell death for treating cancer in acancer patient.

One embodiment of the present invention provides pharmaceuticalcompositions comprising (1) a compound that is impermeable to cellplasma membrane and is capable of interfering, and/or blocking tMPETand/or cell surface respiration, such as WST-1r, WST-3 or their validsubstitute, in combination with (2) the second compound that is capableof suppressing cellular survival signaling, such as NF-κB activities,and/or cellular responses to hypoxia, such as apigenin or its validsubstitute, HIF inhibitors, IKK inhibitors, flavonoids and pUC19 and itsvalid substitutes. Such a pharmaceutical composition may beadministered, in a therapeutically effective amount, in optimizedconcentrations in pharmaceutical acceptable medium, to a patient in needfor the treatment of cancer.

The first compound that, is composed of two functional chemical groups:A) an functional group that is capable of binding to and/or interferingand/or blocking the electron transport process of the tPMET systems,blocking the coupling of oxidative phosphorylation, and/or inhibitingthe tNOX, therefore, to block cell surface respiration and oxygenconsumption; and B) another chemical group or a combination of chemicalgroups that make(s) the entire compound impermeable to cell plasmamembrane and capable of blocking the said compound penetrating the cellplasma membrane and entering the cell. By integrating these functionalgroups into single molecule, the said compound is capable ofinterfering, inhibiting and/or blocking the tPMET, or the oxidativephosphorylation process or the coupling of the oxidative phosphorylationand cell surface respiration specifically on cell surface, but notaffecting the mitochondrial respiration in normal cells.

WST-3 represents such a class of the first compound. It contains adinitrophenol functional group and a chemical group that is impermeableto cell plasma membrane.

FIG. 1 diagrams the chemical structure of WST-3 (Japanese patentJP,2592436,B, 1995), which is composed with a 2,4-Dinitrophenol (DNP),chemical structure as the said functional chemical group and a[1,3-benzenedilsulfonate] and a [4-Iodophenyl] to enhance itshydrophilic feature.

The DNP is an oxidative phosphorylation uncoupler by dissolving in theinner membrane of mitochondria and forms a protonophore, which causedthe protons across the mitochondrial membrane, leading to a rapidconsumption of energy without generating ATP. By integrating the saidDNP with the said second group, the cell plasma impermeable group, itkeeps the DNP from entering the cell, but can only act on the cellplasma membrane. As cancer cells respiration mainly rely on cellsurface, the WST-3 will only blocks the cell surface respiration ofcancer cells, but, will not affect the oxidative phosphorylation inmitochondrial from normal cells, hence, the treatment will be cancerspecific.

The DNP as the said first functional chemical group represents anuncoupler of oxidative phosphorylatoin and may also implicate other waysof blocking tPMET and cell surface respiration. Accordingly, the saidDNP can be substituted by 1) the compounds of oxidative-phsphorylationdecoupling agents comprising: carbonyl cyanide m-chloro phenyl hydrazone(CCCP) and Carbonyl cyanide p-[rifluoromethoxyl]-phenyl-hydrozone(FCCP), SF 6847, salicylanilide S-13, andalpha-(phenylhydrazono)phenylacetonitrile derivatives; and 2)intermediate electron acceptor that direct interact with tPMET,including with no limitation: mPMS and coenzyme Q1; 3) tPMET substrates,such as NADH; 4) the cyanic group (C≡N), such as ferricyanide, andrespiration inhibitors.

The chemical structure of the said second chemical group or combinationof groups that keeps the compound impermeable to cell plasma membranecan be designed and/or produced by a skilled person in the field.Examples include, but not limited to the chemical groups that were usedfor modifying the tetrazolium to form the WSTs, such as the chemicalstructures of the WST-1, WST-3, WST-4, WST-5, WST-8, WST-9, WST-10,WXST-11, XTT, MSN that keep the compound impermeable to the cell plasmamembrane.

Accordingly, the said the first compound is selected from the availablegroups comprising 1) cell plasma membrane impermeable uncoupler WST-3,2) tPMET and/or tNOX inhibitors, including, but not limited tocapsaicin, capsicin pepper vanilloid, green tea catechin,epigallocatechin-3-gallate; 3) the reagents that interfere tPMETactivities including WST-1r and its valid substitutes including but notlimited to WST-3+mPM, WST-4+mPMS, WST-5+mPMS, WST-9+mPMS, WST-10+mPMS,WST-11+mPMS, XTT+mPMS, MSN+mPMS, WST-3+Coenzyme Q1, WST-4+Coenzyme Q1,WST-5+Coenzyme Q1, WST-9+Coenzyme Q1, WST-10+Coenzyme Q1,WST-11+Coenzyme Q1, XTT+Coenzyme Q1, and MSN+Coenzyme Q1; 4) thecompounds that include at least one of the functional groups asdescribed above in the paragraph [00109] and are impermeable to cellplasma membrane that can be designed and produced by a skilled person inthe field.

The said second compound is the one that inhibits cell hepoxiaresponses, which when combined with the first compound, results insynergistic cell death, such as apigenin The said second compound isselected from the groups comprising 1) HIF inhibitors, 2) the flavonoidsand its subclasses such as flavorones, 3inhibitors that inhibit NF-κBactivities, such including, but no limited to IKK inhibitors, 4) plasmidDNA pUC19 [SEQ ID No:1] and its valid substitutes including, but notlimited to at least one of the siRNAs derived from [SEQ ID No: 10, 11,12], means to targeting the genes [SEQ ID No: 2, 3, 4, 5, 14] and theircorresponding gene products [SEQ ID No: 6, 7, 8, 9, 15] to alter theirexpression levels and functional activities including, but not limitedto nucleotide sequences including dominant negative DNA that block thefunction of the corresponding gene products, siRNA, antisnese RNA,antisense oligo, peptides, peptide inhibitors, antibodies, smallmolecule inhibitors.

The said apigenin is a flavone, a subclass of flavonoids, and is amulti-function signal transduction modulator and/or inhibitor to cells.Its function includes, but not limited to induction of p53 activation,suspend cell cycle progression for maintaining genomic stability,inhibiting expression and/or activities of hypoxia induced factor-1(HIF-1), casein kinase II, NF-B, IKK and induction of generation ofreactive oxygen species (ROS) and more.

The second compound and the valid substitutes of apigenin is selectedfrom the groups comprising (1) At least one flavones, include, but notlimited to nature existed flavones, such as: tricin, Luteolin,Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogoninand synthetic flavones, such as: Diosmin, Flavoxate, additionalsubgroups of flavones:flavonols, flavannones, flacanonols, catechins,isoflavones; or

(2) at least one from other subgroups of Flavonoid (Bioflavonoids) andtheir isoforms including naturally existed, artificial modified ketoneisoforms and synthetic compounds including, but not limited toflavonoids, derived from 2-phenylchromen-4-one(2-phenyl-1,4-benzopyrone) structure (examples: quercetin, rutin);isoflavonoids, derived from 3-phenylchromen-4-one(3-phenyl-1,4-benzopyrone) structure; neoflavonoids, derived from4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure, and flavanoidsas a non-ketonepolyhydroxy polyphenol compounds, including: flavanoids,flavan-3-ols and catechins Sample compounds include, but not limited toIsoflavone:Biochanin A, Daidzein, Daidzin, Formononetin, Genistein,Coumestrol, Puerarin; flavan-3-ols: catechins (catechin, epicatechin(EG), epicatechin, gallate (EGC), and epigallocatechin gallate (EGCG));flavonol: myricetin, quercetin, and Kaempferol; Isoflavenes:phenoxodiol; Anthocyanins: Antirrhinin, Chrysanthenin, Malvin,Myrtillin, Oenin Primulin, Protocyanin, Tulipanin; 3-deoxyanthocyanidin:Apigeninidin, Columnidin, Diosmetinidin, Luteolinidin, Tricetinidin;Anthocyanidins: Aurantinidin, Cyanidin, Delphinidin, Europinidin,Luteolinidin, Malvidin, Pelargonidin, Peonidin, Petunidin, Rosinidin;3-Hydroxyflavanones: Dihydrokaempferol, Dihydroquercetin; Flavanones:Eriodictyol, Hesperetin, Homoeriodictyol, Naringenin; Flavonols:Fisetin, Isorhamnetin, Kaempferol, Myricetin, Pachypodol, Quercetin,Rhamnazin, Morin; and their glycoside forms; or

(3) At least one HIF inhibitors and/or inhibition of cellular responsesto hypoxia including, but not limited to: 2,2-dimethybenzopyrancompounds, chetomin, 2-methoxyestradiol (2ME2), PX-478,17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968,camptothecins, NSC 644221,3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, anddecoy oligonucleotides against HIF-1 RX-0047; or

(4) IKK inhibitors are as listed above and following embodiments includecompounds which exhibits IKK inhibitory activity in pharmaceuticallyacceptable medium. The at least one IKK inhibitor may be selected fromcompounds of the group consisting of, without limitation, i) compoundspreviously established to exhibit IKK inhibitory properties including,but not limited to: SPC839 (Signal Pharmaceutical Inc.),Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.),PS1145(Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III,Bristol-Myers Squibb Pharmaceutical Research Institute),SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinonederivative (Bayer), Indolecarboxamide derivative (Aventis Pharma),Benzoimidazole carboxamide derivative (Aventis Pharma),Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation),Imidazolylquinoline-carbxaldehyde semicarbazide derivative (TularkInc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), I B KinaseInhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV[5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKKInhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII(CalBiochem), IKK-2 Inhibitor V(N-(3,5-Bis-trifluoromethylpheny0-5-chloro-2-hydroxybenzamide IMD-0354,CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 InhibitorVIII (ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxypheny0-4-(4-piperidiny0-3-pyridinecarbonitrile,CalBiochem). ii) In a certain embodiment, the group of IKK inhibitorsmay additionally include compounds discovered to have IKK inhibitoryactivity, in accordance, and previously identified as anti-tumor agents,including, but not limited to PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb PharmaceuticalResearch Institute); or

(5) at least one nucleotide sequences [SEQ ID NO:1, 10, 11, 12, 13], andmeans for targeting the genes of polynucleotide sequences [SEQ ID No: 2,3, 4, 5, 14] and peptide sequences [SEQ ID No: 6, 7, 8, 9, 15] toinhibit the expression levels and functional activities of thecorresponding genes by siRNA, antisense RNA, antisense oligo, dominantnegative DNA, peptide, peptide inhibitors, antibodies, small moleculeinhibitors.

2. Pharmaceutical Composition and Method of Wst-3 and ApigeninCombination Treatment For Cancer Therapy

One of the best mode embodiment of the present invention providespharmaceutical compositions comprising (1) at least one Water-solubletetrazolium salts 3(WST-3,2-(4-Iodopheny0-3-(2,4-dinitropheny0-5-(2,4-disulfopheny0-2H-tetrazolium,sodium salt, FIG. 1A) or its valid substitutes in combination with (2)at least one apigenin or its valid substitutes. Such a pharmaceuticalcomposition may be administered, in a therapeutically effective amount,in optimized concentration in pharmaceutical acceptable medium, to apatient in need for the treatment of cancer.

WST-3 is a water soluble tetrazoliums (WST) that were developed byDojindo Inc., whose WSTs have sulfate groups added directly orindirectly to the phenyl ring to improve water-solubility that alsomakes the compound impermeable to cell plasma membrane. Different fromall other WSTs, WST-3 contains a 2,4-dinitrophenol (DNP) group directlylinked to the tetrazolium ring (FIG. 1).

DNP, a cellular metabolic poison, represents a class of six manufacturedchemical compounds that can dissolve in the mitochondria membrane, actsas a proton ionophore, an agent that can shuttle protons (hydrogen ions)across biological membranes, where it uncouples oxidativephosphorylation by carrying protons across the mitochondrial membrane,leading to a rapid consumption of energy without generating ATP. DNPdefeats the proton gradient across mitochondria and chloroplastmembranes, collapsing the proton motive force that the cell uses toproduce most of its ATP chemical energy. Instead of producing ATP, theenergy of the proton gradient is lost as heat. Cells counteract thelowered yields of ATP by oxidizing more stored reserves such ascarbohydrates and fat. DNP has been used as weight loss treatment forburning extra fats. However, it is toxic to the cells by exoughstingcell energy sources.

General structure feature of uncouplers are weak acids comprising thechemical groups: Weakly Acidic Phenols, benzimidazoles,N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylicacids, acyldi-thiocarbazates, cumarines, and aromatic amines.

The chemical structures of representative weakly acidic uncouplers thatare capable of substituting the DNP are selected from the groupscomprising: 5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide(S-13), sodium 2,3,4,5,6-pentachlorophenolate (PCP),4,5,6,7-tetrachloro-2-(trifluoromethyl)-1H-benzimidazole (TTFB),Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid),3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonylcyanide m-chloro phenyl hydrazone (CCCP) and Carbonyl cyanidep-[trifluoromethoxy]-phenyl-hydrazone (FCCP), andalpha-(phenylhydrazono)phenylacetonitrile derivatives.

The incorporating DNP into the water soluble tetrozolium salts thatkeeps the WST-3 impermeable to cell plasma membrane, hence, makes WST3capable of mimicking the DNP effect to act on cell plasma membrane foruncoupling oxidative phosphorylation that interrupts tPMET, but does notaffect mitochondria in normal cells (FIG. 1).

Thus, WST-3 represents classes of compounds that comprises of (1) anactive group that is capable of blocking tPMET and/or oxidativephosphorylation and/or the coupling process between these two processesand (2) the chemical structure that keeps the compound impermeable tocell plasma membrane. In this way such a compound shall be able tospecifically block the tPMET electron transfer and/or oxidativephosphorylation of ADP on cell surface, hence, specifically inhibittPMET and ATP production in cancer cells.

The valid substitutes of WST-3 include, but not limited to the compoundsthat contains the combination of the two said features including (1) theactive group as described above that can block the tPMET and/oroxidative phosphorylation and/or the coupling of the tPMET and theoxidative phosphoryalation process (2) the chemical structure that makesthe resulting compound impermeable to cell plasma membrane as describedabove for the first compound as described in paragraph [0038].

The said Apigenin is a flavonoid and is a multi-function inhibitor tocells. Its function includes, but not limited to induction of p53activation, suspend cell cycle progression to maintain genomicstability, inhibiting expression and/or activities of hypoxia inducedfactor-1 (HIF-1), casein kinase II, NF— B, induction of generation ofROS and more.

The valid substitutes of apigeninare are selected from the groupscomprising: at least one flavones, include, but not limited to natureexisted flavones, such as: Tricin, Luteolin, Tangeritin, Chrysin,6-hydroxyflavone, Baicalein, Scutellarein, Wogonin and syntheticflavones, such as: Diosmin, Flavoxate, additional subgroups of flavones:flavonols, flavannones, flacanonols, catechins, isoflavones; at leastone from other subgroups of Flavonoid or Bioflavonoids and theirisoforms including naturally existed, artificial modified isoforms andsynthetic compounds including, but not limited to flavonoids, derivedfrom 2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure(examples: quercetin, rutin); isoflavonoids, derived from3-phenylchromen-4-one (3-phenyl-1,4-benzopyrone) structure;neoflavonoids, derived from 4-phenylcoumarine (4-phenyl-1,2-benzopyrone)structure, and flavanoids as a non-ketonepolyhydroxy polyphenolcompounds as described in paragraph [00115 and 00114]; at least one HIFinhibitors and/or inhibition of cellular responses to hypoxia including,but not limited to: 2,2-dimethybenzopyran compounds, chetomin,2-methoxyestradiol (2ME2), PX-478,17-N-allylamino-17-demethoxygeldanamycin (17-AAG), EZN-2968,camptothecins, NSC 644221,3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, anddecoy oligonucleotides against HIF-1 RX-0047.

IKK inhibitors are as listed above and following embodiments includecompounds which exhibits IKK inhibitory activity in pharmaceuticallyacceptable medium. The at least one IKK inhibitor may be selected fromcompounds of the group consisting of, without limitation, i) compoundspreviously established to exhibit IKK inhibitory properties including,but not limited to: SPC839 (Signal Pharmaceutical Inc.),Anilino-Pyrimidine Derivative (Signal Pharmaceutical Inc.),PS1145(Millennium Pharmaceutical Inc.), BMS-345541*(IKK inhibitor III,Bristol-Myers Squibb Pharmaceutical Research Institute),SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinonederivative (Bayer), Indolecarboxamide derivative (Aventis Pharma),Benzoimidazole carboxamide derivative (Aventis Pharma),Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation),Imidazolylquinoline-carbxaldehyde semicarbazide derivative (TularkInc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), I B KinaseInhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV[5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKKInhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII(CalBiochem), IKK-2 Inhibitor V(N-(3,5-Bis-trifluoromethylpheny0-5-chloro-2-hydroxybenzamide IMD-0354,CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 InhibitorVIII (ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxypheny0-4-(4-piperidiny0-3-pyridinecarbonitrile,CalBiochem). ii) In a certain embodiment, the group of IKK inhibitorsmay additionally include compounds discovered to have IKK inhibitoryactivity, in accordance, and previously identified as anti-tumor agents,including, but not limited to PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb PharmaceuticalResearch Institute).

(5) at least one nucleotide sequences [SEQ ID NO:1, 10, 11, 12, 13], andmeans for targeting the genes of polynucleotide sequences [SEQ ID No: 2,3, 4, 5, 14] and peptide sequences [SEQ ID No: 6, 7, 8, 9, 15] toinhibit the expression levels and functional activities of thecorresponding genes by siRNA, antisense RNA, antisense oligo, dominantnegative DNA, peptide, peptide inhibitors, antibodies, small moleculeinhibitors.

One embodiment provides methods and a treatment protocol for inducingcancer cell death and tumor suppression to treat cancer in a patient. Inaccordance with this method, it has been discovered that the combinationof WST-3 and/or its valid substitutes with an apigenin and/or its validsubstitutes for synergistic induction of cancer cell death andsuppression of tumor growth.

Accordingly, cancer cells are treated with effective dose(s) of WST-3and/or at least one of its valid substitutes in combination withapigenin and/or at least one of its valid substitutes in pharmaceuticalacceptable medium for effective time period.

The valid substitutes of WST-3 include, but not limited to the compoundsthat contains the combination of the two said features including (1) theactive group as described above that can block the tPMET and/oroxidative phosphorylation and/or the coupling of the tPMET and theoxidative phosphoryalation process (2) the chemical structure that makesthe resulting compound impermeable to cell plasma membrane as describedabove for the first compound as listed in the paragraph [0038].

The suitable active groups that can block the tPMET and oxidativephosphorylation include, but not limited to the DNP group and the cyanogroup.

Suitable as least one valid substitute for apigenin, as listed above,include, but not limited to (1) any other flavonoids and their isoformsincluding naturally existed, artificial modified isoforms and syntheticcompounds, any isoflevens as describd in paragraph [00114 and 00115];(3) inhibitors to HIF-1 and/or any inhibitors to cellular responses tohypoxi as describd in paragraph [00116]; (4) inhibitors to NOXesespecially tNOX as described in paragraph [0035]; (5) inhibitors thatcan mimic one or more of the predetermined apigenin effects.

It is yet another embodiment to treat cancer cells with WST-3 with atleast one apigenin or any of its valid substitutes for both WST-3 andapigenin simultaneously and sequentially in any order for each of theabove and following embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat cancer cells with WST-3 with atleast one IKK inhibitor or all other valid substitutes for both WST-1rand IKK inhibitor simultaneously and sequentially in any order for eachof the above and following embodiments, forming a more preferredembodiment.

The in vitro effective dose of WST-3 may be 50 μM or lower, but can behigher as well.

The effective dose of apigenin under in vitro cell culture may be at1-100 μM.

The WST-3 or at least one of its valid substitutes and apigenin or atleast one of its valid substitutes may be administered to cancer cellsor to cancer patients concurrently, separately and/or sequentially inany order.

Each of the treatment agents may be administrated via oral, intraperitonea injection, intra muscular injection, intra venous injection,intra venous infusion, intra artery infusion, intra artery injection, aswell as via dermal penetration.

The treatment time of WST-3 or at least one of its valid substitutes maybe between pulsed for 30 minutes to 8 hours of initial treatment orcontinuesly.

The treatment time of apigenin or at least one of its valid substitutesmay be last for 15 min to 24 hours consecutively or longer.

In other words, the WST-3 or at least one of its valid substitutes maybe treated first with effective dose for 30 minutes to 4 hours in theabsence of apigenin, then, remove the WST-3 and administer the apigeninor at least one of its valid substitutes to the cancer cells for another4 to 24 hours; or

Alternatively, administering the apigenin or at least one of its validsubstitutes to the cancer cells for another 4 to 24 hours, and then,remove the administer the apigenin or at least one of its validsubstitutes and administering the WST-3 or its valid substitutes tocancer cells for 30 minutes to 4 hours; or

Alternatively, administering the apigenin or at least one of its validsubstitutes and the WST-3 or its valid substitutes to the cancer cellsfor 30 minutes to 4 hours, then remove the treatments and administeringthe apigenin or at least one of its valid substitutes for another 4-24hours, or

Alternatively, administering the apigenin or at least one of its validsubstitutes for 24 hours, then, administering the WST-3 or at least oneof its valid substitutes to the treatment of cancer cells for 30 minutesto 4 hours,

Alternatively, administering of apigenin or at least one of its validsubstitutes and the WST-3 or its valid substitutes can be concurrentlyto the cancer cells for 30 minutes to 4 hours.

Alternatively, administering of apigenin or at least one of its validsubstitutes and the WST-3 or its valid substitutes can be concurrentlyto the cancer cells continuously.

The actual treatment doses of WST-3 and apigenin and the treatment timeof these compounds can be adjusted by a physician or a skilled person.

The preferred embodiment for the treatment is to administer the apigeninor at least one of its valid substitutes and the WST-3 or its validsubstitutes to the cancer cells for 4 hours, then remove the treatmentsand administering the apigenin or at least one of its valid substitutesfor another 24 hours. This is because we have the most date for.

Cancers that may be treated using the combinatorial protocol with WST-3or its valid substitutes in combination with apigenin or its validsubstitutes are carcinomas and sarcomas include, but are not limited tothose carcinomas and sarcomas that may be treated using the presentprotocol include, but are not limited to: cancers of the sqoumas cellcarcinoma, breast, prostate, colorectum, pancreas, cervix, stomach,endometrium, brain, liver, bladder, ovary, testis, head, neck, skin(including melanoma and basal carcinoma), mesothelial lining, esophagus,breast, muscle, connective tissue, lung (including small-cell lungcarcinoma and non-small-cell carcinoma), adrenal gland, thyroid, kidney,or bone; glioblastoma, mesothelioma, renal cell carcinoma, gastriccarcinoma, sarcoma, choriocarcinoma, cutaneous basocellular carcinoma,and testicular seminoma, soft tissue sarcoma, as well as lymphomas andleukemia.

Accordingly, one of the embodiments of this invention provides a methodfor treating cancer in a patient by combination of (1) means of blockingtPMET and/or uncoupling the oxidative-phosphorylation on the cell plasmamembrane with (2) means of inhibiting cellular responses to hypoxia,HIF, NOX, NF-κB activity or mimic one or more of predetermined apigenineffects on cancer cells.

The means to block tPMET and/or uncouple oxidative phosphorylation onthe cell plasma membrane include, but not limited to: the cell plasmamembrane impermeable tMPET oxidative phosphorylation uncoupler or itsvalid substitutes are the compounds that can inhibit the trans plasmamembrane electron transfer process, or the oxidative phosphorylationprocess or the coupling of electron transport and the oxidativephosphorylation and impermeable to cell plasma membrane.

The means of inhibiting cellular responses to hypoxia, HIF, NOX, NF-κBactivity, or mimic one or more of predetermined apigenin effects oncancer cells including, but not limited to treatment with apigenin, orits valid substitutes.

The order of the treatment to cancer cells or cancer patients of themeans of blocking tPMET and/or oxidative phosphorylation on the cellplasma membrane with the means of inhibiting cellular responses tohypoxia, HIF, NOX, NF-κB activity, or mimic one or more of predeterminedapigenin effects on cancer cells can be concurrently or sequentially inany order at effective doses and effective time period for thetreatment.

The present invention also provides additional methods for inducingcancer cell death and suppressing tumor growth in cancer patients. Inaccordance with the present invention, it has been discovered that thecombination of a flavonoid, apigenin, or its valid substitutes, with theWST-3 or the valid substitutes at effective concentration forsynergistic induction of cancer cell death. Accordingly, the presentinvention provides a pharmaceutical composition and protocol for thetreatment of cancer in a patient in need with effective dose comprisingof at least one flavonoid, specifically, apigenin, or its validsubstitutes, with WST-3 or at least one of the valid substitutes of theWST-3 in a pharmaceutical acceptable medium.

Suitable flavonoids include, but not limited to, apigenin and validsubstitutes of apigenin as described above in paragraph [00114-00118] inpharmaceutically acceptable medium.

The valid substitutes of apigenin include the compounds that exhibitinhibitory activity as at least one of the effects of that Apigenin doesin pharmaceutically acceptable medium.

The suitable at least one of the valid substitutes for the WST-3, asnoted herein above in paragraph [00111], include, but are not limited tothe individual components that are comprises the active group asrepresented by DNP and the valid substitutes for tetrazolium salts thatmake the compound impermeable to cell plasma membrane at optimizedconcentrations in pharmaceutically acceptable medium.

The effective concentration of apigenin that were used may varydepending on cell type. The preferred dose is at the range of 1-100 μMin vitro.

For all the above and following embodiments, the effective concentrationof WST-3 and the valid substitutes may vary depending on the individualcomposition and the effective concentration of each of the compositionmay or may not be the same concentration as that in the WST-3 and mayvary from each of the compositions and their valid substitutes andbetween in vitro and in vivo usage. The preferred in vitro concentrationrange for in vitro treatment of WST-3 is 50 μM or lower in apharmaceutical acceptable medium.

In a specific embodiment of the present invention, the administration ofthe WST-3 or at least one valid substitutes of WST-3, the apigenin or atleast one of the valid substitutes of apigenin can be in any type oforder. Specifically, the WST-3 or at least one valid substitutes ofWST-3, and the apigenin or at least one of the valid substitutes ofapigenin may be administered to the cells or patient concurrently orsequentially. In other words, the apigenin or at least one of the validsubstitutes of apigenin or the WST-3 or the at least one substitute ofWST-3 may be administered first, or the WST-3 or at least one validsubstitutes of WST-3, and the apigenin or at least one of the validsubstitutes of apigenin may be administered at the same time. Thepreferred order of the treatment in this invention is to administer theWST-3 or the valid substitutes of WST-3 and the apigenin or the validsubstitutes of apigenin simultaneously and then, after removal of theWST-3, add apigenin again and keep in contact with cells for another 24hours.

In a particular embodiment, the treatment of WST-3 is in contact withcells for 15 minutes to 8 hours. The preferred time is between 30 min to4 hours. The more preferred time is between 2-4 hours. A removal of theWST-3 or its valid substitute's from treatment is required for all theabove and following embodiments to induce programmed cell death of thetreated cells by this method thereof.

Moreover, the present invention provides a method for the treatment ofcancer by administering to a patient, in need thereof, a therapeuticallyeffective dose of at least one of the WST-3 or its valid substitutes andapigenin or at least one of its valid substitutes mentioned above inpharmaceutical acceptable medium.

Cancers that may be treated using the combinatorial protocol with WST-3or its valid substitutes in combination with apigenin include, but arenot limited to Cancers that may be treated using the present protocolinclude, but are not limited to: colorectum, pancreas, cervix, stomach,endometrium, brain, liver, bladder, ovary, testis, head, neck, skin(including melanoma and basal carcinoma), mesothelial lining, whiteesophagus, breast, muscle, connective tissue, lung (including small-celllung carcinoma and non-small-cell carcinoma), adrenal gland, thyroid,kidney, or bone; glioblastoma, mesothelioma, renal cell carcinoma,gastric carcinoma, sarcoma, choriocarcinoma, cutaneous basocellularcarcinoma, and testicular seminoma, leukemia, lymphoma and sarcomas,lymphomas and leukemia.

3. Pharmaceutical Composition and Treatment Method of Combination ofWST-1r and Apigenin for the Treatment of Cancer

One embodiment of the invention provides pharmaceutical compositionscomprising 1) WST-1r or its valid substitutes, which have not previouslybeen established as having an anticancer effect. The WST-1r has beenused as a cell proliferation detection agent, the CellProliferation—WST-1. When WST-1r combined with 2) apigenin, a flavonoid,or its valid substitutes, or an IKK inhibitor, or transfection of Puc19or its valid substitutes synergize the induction of cancer cell death.Such a pharmaceutical composition may be administered, in atherapeutically effective amount, in optimized concentration inphosphate buffered saline or any of the valid pharmaceutical acceptablemedium, to a patient in need for the treatment of cancer.

The afficacy of the said anticancer treatment immediate above wassynergized by combination use of WST-1r and its valid substitutes whichhave not previously been established. The Cell Proliferation WST-1r iscomposed of a tetrazolium salt, WST-1c (WST-1, Ishiyam M, et al BiolPharm Bull 1996, 19:1515-20; Berridge M V, et al Biotechnology AnnualReview, Vol. II: 127-152, 2005), and an IEA, mPMS, (Berridge M V, et alBiotechnology Annual Review, Vol. II: 127-152, 2005) diluted in phosphorbuffered saline. WST-1r has also been used for measuring tPMET activity.Treatment with WST-1r enhanced cell respiration. When the WST-1rtreatment was withdraw following the treatment and in combination ofinhibiting HIF by apigenin or any of its valid substitutes resulted insynergized cancer cell death. In the present invention, WST-1r is usedas a drug for a combination treatment for cancer therapy.

In accordance, the active gradient of WST-1r for the treatment of cancercan be either the WST-1c or the mPMS or the combination of the twocomponents in optimized concentration and optimized ratio. The WST-1rthat as described herein above and there after represents a group ofchemical compound or mixture of combinations of a water solubletetrazolium salt and an IEA that are capable of interacting with and/orinterfering to tPMET, and/or capable of inducing reactive oxygen species(ROS) generation.

The valid substitutes of WST-1c include, but not limited to other WST,including, but not limited to WST-3, WST-4, WST-5, WST-9, WST-10,WST-11, MSN and XTT at optimized concentration in a pharmaceuticalacceptable medium.

The valid substitutes of mPMS include, other IEAs, examples may be as,but not limited to coenzyme Q1 (Berridge M V, et al Biotechnology AnnualReview, Vol. II: 127-152, 2005) at optimized concentration in apharmaceutical acceptable medium.

The WST-1r includes compositions of at least one WST, WST-1c, and atlease one IEA, mPMS in optimized concentration and ratio in apharmaceutical acceptable medium.

The valid substitute of WST-1r includes, but not limited to (1) thecombination of at least one WST with at least one IEA. Examples as, butnot limited to: WST-1+mPMS, WST-3+mPMS, WST-4+mPMS WST-5+mPMS,WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, MSN+mPMS XTT+mMS, WST-1+coenzymeQ1, WST-3+coenzyme Q1, WST-4+coenzyme Q1 WST-5+coenzyme Q1,WST-9+coenzyme Q1, WST-10+coenzyme Q1, WST-11+coenzyme Q1, MSN+coenzymeQ1 XTT+coenzyme Q1; (2) at least one of the WST, such as, with nolimitation, WST-3; (3) at least one IEA, such as, with no limitation,mPMS and coenzyme Q1 at optimized concentration in a pharmaceuticallyacceptable medium.

The Apigenin herein represents the second molecule of this combinationcomposition. The valid substitutes of apigenin are selected from thegroups comprising: 1) at least one flavone as listed above in paragraph[00114], 2) at least one flavonoids or isoflavonoids as listed above inparagraph [00115]; 3) at least one HIF inhibitors as described above inparagraph [00116], 3) at least one IKK inhibitors as described above inparagraph [00117], 4) at least one nucleotide sequences [SEQ ID NO:1,10, 11, 12, 13], and means for targeting the genes of polynucleotidesequences [SEQ ID No: 2, 3, 4, 5, 14] and peptide sequences [SEQ ID No:6, 7, 8, 9, 15] as listed above in paragraph [00118].

It is yet another embodiment to treat cancer cells with WST-1r andapigenin, a flavonoids or all other valid substitutes for both WST-1rand apigenin simultaneously and sequentially in any order for each ofthe above and following embodiments, forming a more preferredembodiment.

It is yet another embodiment to treat cancer cells with WST-1r with atleast one IKK inhibitor or all other valid substitutes for both WST-1rand IKK inhibitor simultaneously and sequentially in any order for eachof the above and following embodiments, forming a more preferredembodiment.

It is yet another embodiment to treat cancer cells with (1) the DNAtransfection, or IFN, or siRNA transfection or all other validsubstitutes and, then, (2) one of the IKK, or CK2 or GSK3 inhibitorsand, treat with WST-1r simultaneously or sequentially in any order foreach of the above and following embodiments, forming a more preferredembodiment.

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, treat with electroncoupling reagent of the WST-1r simultaneously or sequentially in anyorder for each of the above and following embodiments, forming a morepreferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, simultaneously orsequentially in any order treat with all the remaining subcomponent ofthe WST-1r for each of the above and following embodiments, forming amore preferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, simultaneously orsequentially in any order treat with any valid substitution for WST-1rfor each of the above and following embodiments, forming a morepreferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, simultaneously orsequentially in any order treat with any valid substitution for WST-1cfor each of the above and following embodiments, forming a morepreferred embodiment

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, simultaneously orsequentially in any order treat with any valid substitution for electroncoupling reagent of the WST-1r, such as mPMS, for each of the above andfollowing embodiments, forming a more preferred embodiment.

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, simultaneously orsequentially in any order treat with any valid substitution for theremaining subcomponent of the WST-1r for each of the above and followingembodiments, forming a more preferred embodiment.

It is yet another embodiment to treat with (1) the DNA transfection, orIFN, or siRNA transfection or all other valid substitutes and, then, (2)one of the IKK, or CK2 or GSK3 inhibitors and, treat with WST-1rsimultaneously or sequentially in any order treat with any validsubstitution as any type of combination of the valid substitutes and thesubcomponent of the WST-1r for each of the above and followingembodiments, forming a more preferred embodiment.

Moreover, the present descriptions provide pharmaceutical compositionsand methods for the treatment of cancer by administering to a patient,in need thereof, a therapeutically effective amount of at least one ofthe WST-1r component or its valid substitutes mentioned immediatelyabove.

The optimized concentration may or may not be the same concentration asthat of the Cell Proliferation WST-1 reagent and may vary from each ofthe compositions and their valid substitutes and between in vitro and invivo usage. The preferred optimized in vitro WST-1r, WST-3+mPMS,WST-4+mPMS and WST-3 are the most preferred embodiment because they werethe component for which we have the most valid data.

Moreover, the present description provides a method for the treatment ofcancer by administering to a patient, in need thereof, a therapeuticallyeffective amount of at least one of the WST-1r component or its validsubstitutes mentioned immediately above.

In a particular embodiment, the preferred treatment of WST-1r is incontact with cells for at lease 15 minutes or longer. The more preferredtreatment time for WST-1r is between 30 min to 4 hours. The even morepreferred treatment time for WST-1r is between 2-4 hours.

Each of the treatment agents may be administrated via oral, intraperitonea injection, intra muscular injection, intra venous injection,intra venous infusion, intra artery infusion, intra artery injection, aswell as via dermal penetration.

Cancers that may be treated using the present protocol include, but arenot limited to: carcinoma derived from prostate, colorectum, pancreas,cervix, stomach, endometrium, brain, liver, bladder, ovary, testis,head, neck, skin (including melanoma and basal carcinoma), mesotheliallining, white esophagus, breast, muscle, connective tissue, lung(including small-cell lung carcinoma and non-small-cell carcinoma),adrenal gland, thyroid, kidney, or bone; glioblastoma, mesothelioma,renal cell carcinoma, gastric carcinoma, sarcoma, choriocarcinoma,cutaneous basocellular carcinoma, and testicular seminoma, leukemia,lymphoma and sarcomas.

4. Combinatorial Therapies with Inhibitors and WST-1r for the Treatmentof Cancer

The present description provides additional methods for inducing cancercell death for the treatment of cancer for a patient in need. Inaccordance, it has been discovered that the combination of pUC19 DNAtransfection and/or its valid substitutes with an IKK inhibitor plusWST-1r or its valid substitutes for synergistic inducing cancer celldeath. Accordingly, the present description provides a pharmaceuticalcomposition and protocol for the treatment of cancer in a patientcomprising at lease pUC19 DNA transfection or its valid substitutes incombination with at least one IKK inhibitor and WST-1r or at least oneof the valid substitutes of the WST-1r. Also provided is a method fortreating cancer in a patient by IFN in combination with administering aneffective amount of at least one IKK inhibitor and WST-1r or at leastone of the valid substitutes of the WST-1r. Also provided is a methodfor treating cancer in a patient by transfection of the cells with siRNAin combination with administering an effective amount of at least oneIKK inhibitor and WST-1r or at least one of the valid substitutes of theWST-1r.

The DNA transfection may be substituted by (i) administering a suitabledose of at least one IFN, or (ii) transfection of at least one specificsiRNA targeting at least one of the target transcripts as describedpreviously in this description, or (iii) chemical compounds or smallmolecule inhibitors that targets at least one of the target genes and/orits gene products as described previously in this description, or (iv)antibodies targeting at least one of the target genes products asdescribed previously in this description, (v) anti-sense RNAs targetingat least one of the target transcripts as described previously in thisdescription, (vi) shRNAs targeting at least one of the targettranscripts as described previously in this description, (vii)anti-sense oligos targeting at least one of the target transcripts asdescribed previously in this description, (viii) A dominant negative DNAvector targeting at least one of the target genes as describedpreviously in this description, (ix) peptides targeting at least one ofthe target genes products as described previously in this description.

The target genes are, but not limited to, (1) Homo sapiens transientreceptor potential cation channel, subfamily C, member 6(TRPC6, GeneID:7225), mRNA (NM_(—)004621.3) synonyms: TRP6, FSGS2, FLJ11098 (SEQ ID #2,#6), (2) Homo sapiens SH3 and PX domains 2B (SH3PXD2B), mRNA (.(SH3PXD2B, GeneID: 285590), mRNA (NM_(—)001017995) synonyms: HOFI;FLJ20831; KIAAl295 (SEQ ID #3, #7), (3) Homo sapiens membrane associatedguanylate kinase, WW and PDZ domain containing 3 (MAGIKK, GeneID:260425), transcript variant 2, mRNA (NM_(—)152900.1) synonyms: MAGI-3,MGC163281 (SEQ ID #4, #8), and (4) the Homo sapiens transmembraneprotein 182 (TMEM182, GeneID: 130827), mRNA (NM_(—)144632.2) (SEQ ID #5,#9).

The gene products include, but not limited to, the transcripts fromthese genes and proteins above.

The siRNA sequences and the targets of the siRNA sequences may alsoinclude the human genomic sequences that flanking the genes as listed inthe attached file entitled: “NCBI Blast-pUC19-Human-Transcripts andgenome (2686 letters)”, “NCBI Blast_siRNA2 Nucleotide sequence (24letters)” and “NCBI Blast_pcDNA3 Nucleotide sequence (5448 letters)”.NCBI Blast-pUC19-Human-Transcripts and genome.

The at least one IFN may be selected from the subfamily of type I IFNincluding, but not limited to: IFN A, IFN B, IFN C, IFN D, IFN F, IFN G,IFN H, IFN I, IFN J, IFN K, IFN 4b, IFN WA, and IFN.

The effective concentration of IFN that were used for treating cancercells was 10 unit/ml or lower for each IFN used.

Suitable IKK inhibitors include any compound which exhibits IKKinhibitory activity.

The at least one IKK inhibitor may be selected from compounds of thegroup consisting of, without limitation, i) compounds previouslyestablished to exhibit IKK inhibitory properties including, but notlimited to: SPC839 (Signal Pharmaceutical Inc.), Anilino-PyrimidineDerivative (Signal Pharmaceutical Inc.), PS1145(MillenniumPharmaceutical Inc.), BMS-345541*(IKK inhibitor III, Bristol-MyersSquibb Pharmaceutical Research Institute), SC-514*(Smithkilne BeechamCorp.), Amino-imidazolecarboxamide derivative (Smithkilne BeechamCorp.), Ureudo-thiophenecarboxamide derivatives (AstraZeneca),Diarylpybidine derivative (Bayer), Pyridooxazinone derivative (Bayer),Indolecarboxamide derivative (Aventis Pharma), Benzoimidazolecarboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinolinederivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehydesemicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), I B Kinase Inhibitor Peptide (CalBiochem), IKK-2 InhibitorIV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem),IKK Inhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII(CalBiochem), IKK-2 Inhibitor V(N-(3,5-Bis-trifluoromethylpheny0-5-chloro-2-hydroxybenzamide IMD-0354,CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 InhibitorVIII (ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxypheny0-4-(4-piperidiny0-3-pyridinecarbonitrile,CalBiochem). ii) In a certain embodiment, the group of IKK inhibitorsmay additionally include compounds discovered to have IKK inhibitoryactivity, in accordance, and previously identified as anti-tumor agents,including, but not limited to PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb PharmaceuticalResearch Institute).

Suitable WST-1r and the at least one of the valid substitutes of theWST-1r, as noted herein above, include, but are not limited to to (1)the combination of at least one WST with at least one IEA. Examples as,but not limited to: WST-1+mPMS, WST-3+mPMS, WST-4+mPMS WST-5+mPMS,WST-9+mPMS, WST-10+mPMS, WST-11+mPMS, MSN+mPMS XTT+mMS, WST-1+coenzymeQ1, WST-3+coenzyme Q1, WST-4+coenzyme Q1 WST-5+coenzyme Q1,WST-9+coenzyme Q1, WST-10+coenzyme Q1, WST-11+coenzyme Q1, MSN+coenzymeQ1 XTT+coenzyme Q1; (2) at least one of the WST, such as, with nolimitation, WST-3; (3) at least one IEA, such as, with no limitation,mPMS and coenzyme Q1 at optimized concentration in a pharmaceuticallyacceptable medium.

In a specific embodiment, the preferred order of treatment is toadminister the pUC19 DNA transfection or its valid substitutes, at leastone IKK inhibitor and WST-1r or at least one of the valid substitutes ofWST-1r concurrently and/or sequentially in any type of order. However,the pUC19 DNA transfection or IFN treatment, or siRNA transfection orits other valid substitutes, at least one IKK inhibitor and the WST-1ror the at least one valid substitutes of WST-1r may be administered tothe cells or patient concurrently or sequentially. In other words, thepUC19 DNA transfection may be treated first, the at least one IKKinhibitor may be administered first, the WST-1r or the at least onesubstitute of WST-1r may be administered first, or the pUC19 DNAtransfection, the at least one IKK inhibitor and the at least onesubstitute of WST-1r may be administered at the same time. Additionally,when the pUC19 DNA transfection is replaced by siRNA transfection, IFNadministration, or small molecule targeting the target genes asdescribed in this description above, in combination with at least oneIKK inhibitor and WST-1r or at least one valid substitute of WST-1r isused, the compounds may be administered in any order.

Cancers that may be treated using the present combinatorial protocol arecarcinomas and sarcomas, lymphomars and leukemia include, but are notlimited to those cancers described herein above in paragraph [00164].However, the suitable cancer cells and tumors that may be moresusceptible to this treatment are those with aberrant NF-κB activities.

The present description also provides additional methods for inducingcancer cell death and suppressing tumor in cancer patients. Inaccordance, it has been discovered that the combination of a flavonoid,apigenin, or its valid substitutes, or an IKK inhibitor at effectiveconcentration with the WST-1r or the valid substitutes at effectiveconcentration for synergistic induction of cancer cell death.Accordingly, the present description provides a pharmaceuticalcomposition and protocol for the treatment of cancer in a patient inneed with effective dose comprising of at least one flavonoid,preferably, apigenin, or its valid substitutes, or an IKK inhibitor withWST-1r or at least one of the valid substitutes of the WST-1r in apharmaceutical acceptable medium.

A removal of the treatment is required for all the above and followingembodiments to induce programmed cell death of the treated cells by thismethod.

Suitable flavonoids include, but not limited to, apigenin, theflavonoids, and valid substitutes of apigenin as described above inparagraph [00114 and 00115] in pharmaceutically acceptable medium.

The valid substitutes of apigenin are selected from the groupscomprising The second compound and the valid substitutes of apigenin isselected from the groups comprising (1) At least one flavones, include,but not limited to nature existed flavones, such as: Luteolin,Tangeritin, Chrysin, 6-hydroxyflavone, Baicalein, Scutellarein, Wogoninand synthetic flavones, such as: Diosmin, Flavoxate, additionalsubgroups of flavones:flavonols, flavannones, flacanonols, catechins,isoflavones in paragraph [00114]; or (2) at least one from othersubgroups of Flavonoid or Bioflavonoids and their isoforms includingnaturally existed, artificial modified isoforms and synthetic compoundsincluding, but not limited to flavonoids, derived from2-phenylchromen-4-one (2-phenyl-1,4-benzopyrone) structure (examples:quercetin, rutin); isoflavonoids, derived from 3-phenylchromen-4-one(3-phenyl-1,4-benzopyrone) structure; neoflavonoids, derived from4-phenylcoumarine (4-phenyl-1,2-benzopyrone) structure, and flavanoidsas a non-ketonepolyhydroxy polyphenol compounds as described above inparagraph [00114 and 00115]; or (3) At least one HIF inhibitors and/orinhibition of cellular responses to hypoxia including, but not limitedto: 2,2-dimethybenzopyran compounds, chetomin, 2-methoxyestradiol(2ME2), PX-478, 17-N-allylamino-17-demethoxygeldanamycin (17-AAG),EZN-2968, camptothecins, NSC 644221,3-(5′-hydroxymethyl-2′-furyl)-1-benzylindazole (YC-1), rapamycin, anddecoy oligonucleotides against HIF-1 RX-0047; as described above inparagraph [00116] in effective doses and in pharmaceutically acceptablemedium.

Suitable IKK inhibitors are as listed above and following embodimentsinclude any compound which exhibits IKK inhibitory activity inpharmaceutically acceptable medium. The at least one IKK inhibitor maybe selected from compounds of the group consisting of, withoutlimitation, i) compounds previously established to exhibit IKKinhibitory properties including, but not limited to: SPC839 (SignalPharmaceutical Inc.), Anilino-Pyrimidine Derivative (SignalPharmaceutical Inc.), PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb PharmaceuticalResearch Institute), SC-514*(Smithkilne Beecham Corp.),Amino-imidazolecarboxamide derivative (Smithkilne Beecham Corp.),Ureudo-thiophenecarboxamide derivatives (AstraZeneca), Diarylpybidinederivative (Bayer), Pyridooxazinone derivative (Bayer),Indolecarboxamide derivative (Aventis Pharma), Benzoimidazolecarboxamide derivative (Aventis Pharma), Pyrazolo[4,3-c]quinolinederivative (Pharmacia Corporation), Imidazolylquinoline-carbxaldehydesemicarbazide derivative (Tulark Inc.), Pyridyl Cyanoguanidine derivate(Leo Pharma), I B Kinase Inhibitor Peptide (CalBiochem), IKK-2 InhibitorIV [5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem),IKK Inhibitor II (Wedelolactone (CalBiochem), IKK Inhibitor VII(CalBiochem), IKK-2 Inhibitor V(N-(3,5-Bis-trifluoromethylpheny0-5-chloro-2-hydroxybenzamide IMD-0354,CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide, CalBiochem), IKK-2 InhibitorVIII (ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxypheny0-4-(4-piperidiny0-3-pyridinecarbonitrile,CalBiochem). ii) In a certain embodiment, the group of IKK inhibitorsmay additionally include compounds discovered to have IKK inhibitoryactivity, in accordance, and previously identified as anti-tumor agents,including, but not limited to PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(IKK inhibitor III, Bristol-Myers Squibb PharmaceuticalResearch Institute).

Suitable WST-1r and the at least one of the valid substitutes of theWST-1r, as noted herein above, include, but are not limited to WST-1rand each of the individual components, the WST-1c anf mPMS, that arecomprises the WST-1r, the valid substitutes for WST and that for IEA ofthe WST-1r and all possible combination among these valid substitutes ofWST-1 and mPMS or the combination of these valid substitutes and theindividual component of the WST-1r, the WST and IEA as described abovein paragraph [00171], at optimized concentrations in pharmaceuticallyacceptable medium.

The effective concentration of apigenin that were used may varydepending on cell type. For all the above and following embodiments, theeffective concentration of WST-1r and the valid substitutes may varydepending on the individual composition and the effective concentrationof each of the composition may or may not be the same concentration asthat in the Cell Proliferation WST-1 reagent and may vary from each ofthe compositions and their valid substitutes and between in vitro and invivo usage.

In a specific embodiment, the administration of the WST-1r or at leastone valid substitutes of WST-1r, the apigenin, the flavonoid or at leastone of the valid substitutes of apigenin or the at least one IKKinhibitor can be in any type of order. Specifically, the WST-1r or atleast one valid substitutes of WST-1r, and the apigenin or at least oneof the valid substitutes of apigenin or the at least one IKK inhibitormay be administered to the cells or patient concurrently orsequentially. In other words, the apigenin or at least one of the validsubstitutes of apigenin or the at least one IKK inhibitor may beadministered first, the WST-1r or the at least one substitute of WST-1rmay be administered first, or the WST-1r or at least one validsubstitutes of WST-1r, and the apigenin or at least one of the validsubstitutes of apigenin or the at least one IKK inhibitor may beadministered at the same time. The preferred order of the treatment isto administer the WST-1r or the valid substitutes of WST-1r and theapigenin or the valid substitutes of apigenin or at least one IKKinhibitor simultaneously and then, after removal of the WST-1r, addapigenin or IKK inhibitor again and keep in contact with cells foranother 24 hours.

In a particular embodiment, the in vitro treatment of WST-1r is incontact with cells for at least 15 minutes or longer The preferred timeis between 30 min to 4 hours. The more preferred time is between 2-4hours. A removal of the WST-1r or its valid substitute's from treatmentis required for all the above and following embodiments to induceprogrammed cell death of the treated cells by this method thereof.

Moreover, the present description provides a method for the treatment ofcancer by administering to a patient, in need thereof, a therapeuticallyeffective dose of at least one of the WST-1r or its valid substitutesand apigenin or at least one of its valid substitutes as described abovein pharmaceutical acceptable medium.

Also, the present description provides a method for the treatment ofcancer by administering to a patient, in need thereof, a therapeuticallyeffective dose of at least one of the WST-1r or its valid substitutesand at least one IKK inhibitor mentioned above in pharmaceuticalacceptable medium.

Cancers that may be treated using the combinatorial protocol with WST-1ror its valid substitutes in combination with apigenin include, but arenot limited to those carcinomas and sarcomas that may be treated usingthe present protocol include, but are not limited to: cancers of theprostate, colorectum, pancreas, cervix, stomach, endometrium, brain,liver, bladder, ovary, testis, head, neck, skin (including melanoma andbasal carcinoma), mesothelial lining, esophagus, breast, muscle,connective tissue, lung (including small-cell lung carcinoma andnon-small-cell carcinoma), adrenal gland, thyroid, kidney, or bone;glioblastoma, mesothelioma, renal cell carcinoma, gastric carcinoma,sarcoma, choriocarcinoma, cutaneous basocellular carcinoma, lymphoma,leukemia and testicular seminoma, soft tissue sacoma.

5. Other Compositions and Methods for Enhance and Synergize theTreatment of Cancer

The present description provides additional methods for synergisticinhibition of NF-κB activity in cancer cells. In accordance, it has alsobeen discovered that the pUC19 DNA transfection may also synergize theinhibition of NF-κB activity in cancer cells when both IKK1-KA andIKK2-KA kinase dead dominant negative vector were used simultaneously.This inhibitory effect can be further enhanced by the combination ofadditional treatment of WST-1r or at least one of the valid substitutesfor WST-1r.

Accordingly, pUC19 DNA trasnfection may be substituted by treating thecells or a mammal with (i) administering a suitable dose of at least oneIFN, or (ii) transfection of at least one specific siRNA or shRNAtargeting at least one of the target transcripts as described previouslyin this specification, or (iii) small molecule inhibitors that targetsat least one of the target genes products as described previously inthis specification, or (iv) antibodies and peptide inhibitors targetingat least one of the target genes products as described previously inthis specification, (v) anti-sense RNA targeting at least one of thetarget transcripts as described previously in this specification, (vi)anti-sense oligo targeting at least one of the target gene's transcriptsas described previously in this specification in combination with thetreatment of at least one IKK inhibitors that can inhibit both IKK1 andIKK2 kinase activities.

The at least one IFN may be selected from the subfamily of IFNincluding, but not limited to: IFN A, IFN B, IFN C, IFN D, IFN F, IFN G,IFN H, IFN I, IFN J, IFN K, IFN 4b, IFN WA, IFN IFN or IL-6.

The transcripts, and proteins as the targets of the siRNA, shRNA, smallmolecule inhibitor, peptide inhibitor, antibody, anti-sense RNA,anti-sense oligo, and antibody are, but not limited to, (1) Homo sapienstransient receptor potential cation channel, subfamily C, member6(TRPC6, SEQ ID 2, 6), (2) Homo sapiens SH3 and PX domains 2B (SH3PXD2B,SeQ ID #3, #7), (3) Homo sapiens membrane associated guanylate kinase,WW and PDZ domain containing 3 (MAGIKK, SeQ ID #4, #8), (4) the Homosapiens transmembrane protein 182 (TMEM182, SeQ ID #5, #9) and (5) theC6orf108 (Seq ID #14, #15).

Suitable WST-1r and the at least one of the valid substitutes of theWST-1r, as noted herein above, include, but are not limited to WST-1rand each of the individual tetrazolium components that are comprises theWST-1r, the valid substitutes of each component of the WST-1r and anytype of combination among these valid substitutes or the combinationamong these valid substitutes and the individual component of the WST-1and mPMS.

The at least one IKK inhibitor may be selected from compounds of thegroup consisting of: i) compounds previously established to exhibit IKKinhibitory properties including, but not limited to: SPC839 (SignalPharmaceutical Inc.), Anilino-Pyrimidine Derivative (SignalPharmaceutical Inc.), PS1145(Millennium Pharmaceutical Inc.),BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute),SC-514*(Smithkilne Beecham Corp.), Amino-imidazolecarboxamide derivative(Smithkilne Beecham Corp.), Ureudo-thiophenecarboxamide derivatives(AstraZeneca), Diarylpybidine derivative (Bayer), Pyridooxazinonederivative (Bayer), Indolecarboxamide derivative (Aventis Pharma),Benzoimidazole carboxamide derivative (Aventis Pharma),Pyrazolo[4,3-c]quinoline derivative (Pharmacia Corporation),Imidazolylquinoline-carbxaldehyde semicarbazide derivative (TularkInc.), Pyridyl Cyanoguanidine derivate (Leo Pharma), IkB KinaseInhibitor Peptide (CalBiochem), IKK-2 Inhibitor IV[5-(p-Fluorophenyl)-2-ureido]thiophene-3-carboxamide (CalBiochem), IKKInhibitor II, Wedelolactone (CalBiochem), IKK Inhibitor VII K InhibitorVII (CalBiochem), IKK-2 Inhibitor VN-(3,5-Bis-trifluoromethylpheny0-5-chloro-2-hydroxybenzamideIMD-0354(CalBiochem), IKK-2 Inhibitor VI(5-Phenyl-2-ureido)thiophene-3-carboxamide (CalBiochem), IKK-2 InhibitorVIII ACHP2-Amino-6-(2-(cyclopropylmethoxy)-6-hydroxypheny0-4-(4-piperidiny0-3-pyridinecarbonitrile(CalBiochem). In a certain embodiment, the group of IKK inhibitors mayadditionally include compounds discovered to have IKK inhibitoryactivity, in accordance, and previously identified as anti-tumor agents,including, but not limited to PS 1145 (Millennium Pharmaceutical Inc.),BMS-345541*(Bristol-Myers Squibb Pharmaceutical Research Institute). Thepreferred IKK inhibitors are the IKK inhibitors that can inhibit bothIKK1 and IKK2 kinase activities.

The present description provides additional medical use for inducingcancer cell death and tumor suppression. In accordance, it has beendiscovered that the combination of a GSK3 inhibitor with a CK2 inhibitorin combination with WST-1r or at least one of the valid substitutes forWST-1r act synergistically to suppress tumor growth. Accordingly, thepresent description provides a pharmaceutical composition for thetreatment of cancer in a subset of cancer cells and/or in a patientcomprising at least one GSK3 inhibitor, at least one CK2 inhibitor andWST-1r or the at least one of the valid substitutes for WST-1 in apharmaceutically acceptable carrier. Also provided is a method fortreating cancer in a patient by administering an effective amount of atleast one GSK3 inhibitor in combination with at least one CK2 inhibitor.Suitable GSK3 inhibitors include any compound which exhibits GSK3inhibitory activity, for example, LiCl. Suitable CK2 inhibitors,include, but are not limited to: Apigenin

The at least one CK2 inhibitor may be selected from compounds of thegroup comprising, but not limited to: TBB, TBBz, emodin, CK2 inhibitorIII (sigma).

Suitable WST-1r and the at least one of the valid substitutes of theWST-1r, as noted herein above, include, but are not limited to WST-1rand each of the individual components that comprises the WST-1r, thevalid substitutes of each component of the WST-1r and any type ofcombination among these valid substitutes or the combination among thesevalid substitutes and the individual component of the WST-1r.

In a specific embodiment, the at least one GSK3 inhibitor and at leastone CK2 inhibitor may be administered to the cancer cells or patientconcurrently or sequentially. In other words, the at least one GSK3inhibitor may be administered first, the at least one CK2 inhibitor maybe administered first, or the at least one GSK3 inhibitor and the atleast one CK2 inhibitor may be administered at the same time.Additionally, when more than one GSK3 inhibitor and/or CK2 inhibitor areused, the compounds may be administered in any order.

Cancer cells that may be treated using the present combinatorialprotocol include, but are not limited to UM-SCC-6 cells. Cancers thatmay be treated using the present combinational protocol include, but arenot limited to, those cancers described herein.

The present description provides additional medical use for enhancing orsynergizing the efficacy effects of chemotherapy drugs for the treatmentof cancer. In accordance, it has also been discovered that the Puc19 DNAtransfection also synergizes suppression of tumor growth and promotescancer cell death. Accordingly, the present description provides apharmaceutical composition for the treatment of cancer in a patientcomprising puc19 DNA transfection or at least one of its validsubstitutes and at least one chemotherapeutic agent. This induction ofcancer cell death effect may be further enhanced by additionalcombination with WST-1r or at least one of the valid substitutes ofWST-1r in a pharmaceutically acceptable carrier. Also provided is amethod for treating cancer cells or cancer in a patient by administeringan effective dose of at least one DNA transfection or at least one ofthe valid substitutes for DNA transfection in combination with at leastone chemotherapeutic agent. In a preferred embodiment, the preferred DNAfor transfection is pUC19 DNA cloning vector as described previous inthis application (Sequence #1).

The at least one valid substitute for the pUC19 DNA transfection mayinclude, but not limited to, (i) administering a suitable dose of atleast one IFN, or (ii) transfection of at least one specific siRNAtargeting at least one of the target transcripts as described previouslyin this specification, or (iii) at least one chemical compounds or smallmolecule inhibitors that targets at least one of the target genes and/orits gene products as described previously in this specification, or (iv)at lease one antibody targeting at least one of the target genesproducts as described previously in this specification, or (v)anti-sense RNA targeting at least one of the target transcripts asdescribed previously in this specification, (vi) shRNA targeting atleast one of the target transcripts as described previously in thisspecification, (vii) anti-sense oligo targeting at least one of thetarget transcripts as described previously in this specification, (viii)A dominant negative DNA vector targeting at least one of the targetgenes as described previously in this specification, (ix) peptidestargeting at least one of the target genes products as describedpreviously in this specification.

Suitable IFN may be selected from any IFN subfamily members, whichinclude, but not limited to, IFN A, IFN B, IFN C, IFN D, IFN F, IFN G,IFN H, IFN I, IFN J, IFN K, IFN 4b, WA, IFN, IFN and Interlukine-6(IL-6). In a preferred embodiment, the preferred IFN are subfamilymembers of IFN, IFN. The effective concentration of IFN is 10 unit/ml orlower for each IFN.

The target genes to be targeted by the at least one chemical compoundsor small molecule inhibitors, at least one specific siRNA, shRNA,anti-sense RNA, anti-sense oligo, dominant negative DNA vector, at leastone peptide, at lease one antibody, at least one inhibitor are, but notlimited to, (1) TRPC6, (SEQ ID #2, #6), (2) SH3PXD2B, (SEQ ID #3, #7),(3) MAGIKK, (SEQ ID #4, #8), (4) TMEM182, (SEQ ID #5, #9), and (5)C6orf108 (Seq ID #14, #15).

The gene products include, but not limited to, the nucleotide sequenceof the transcripts from the gene and amino acid sequence of the proteinthat derived from these genes.

The siRNA and or shRNA sequences and the targets of the siRNA sequencesmay also include the nucleotide sequence that mapped to the humangenomic sequences that flanking the genes as listed in the attached file“NCBI Blast-pUC19-Human-Transcripts and genome (2686 letters)” and “NCBIBlast_siRNA2 Nucleotide sequence (24 letters)”.

Accordingly, Suitable siRNAs include siRNA1 (SEQ ID #10), siRNA 2(SEQ ID#11), and siRNA 3(SEQ ID #12) as described previous in thisspecification and all the potential siRNAs that may be derived frompUC19 DNA sequence that mapped to human genome and/or transcripts inshort pieces (10-100 by and more). These nucleotide sequences and theircorresponding genes are listed in the attached file “NCBIBlast-pUC19-Human-Transcripts and genome (2686 letters)” and “NCBIBlast_siRNA2 Nucleotide sequence (24 letters)”. As in general, thesesiRNA sequences can be vary up to 40% from the exact sequences of thegene. Additionally, the function of these siRNAs can be substituted byany of the siRNA and/or shRNA that mapped to other part sequences of thecorresponding target gene, small molecule inhibitors, peptideinhibitors, antibodies, anti-sense RNAs, anti-sense oligos and dominantnegative DNA vectors that can effectively target the gene products astargets, which are the target of the siRNAs as described above in thisparagraph and are include, but not limited to, (1) TRPC6, (SEQ ID #2,#6), (2) SH3PXD2B, (SEQ ID #3, #7), (3) MAGIKK, (SEQ ID #4, #8), (4)TMEM182, (SEQ ID #5, #9), and (5) C6orf108 (Seq ID #14, #15).

The WST-1r or at least one of the valid substitutes of WST-1r, as notedherein above, include, but are not limited to WST-1r and each of theindividual components that are comprises the WST-1r, the validsubstitutes of each component of the WST-1r and any type of combinationamong these valid substitutes or the combination among these validsubstitutes and the individual component of the WST-1r.

Suitable chemotherapeutic agents include, but are not limited to:paclitaxel (Taxol®), cisplatin, docetaxol, carboplatin, vincristine,vinblastine, methotrexate, cyclophosphamide, CPT-11, 5-fluorouracil(5-FU), gemcitabine, estramustine, carmustine, adriamycin (doxorubicin),etoposide, arsenic trioxide, irinotecan, and epothilone derivatives. Thepreferred chemotherapeutic agents are paclitaxel (Taxol®), cisplatin,5-fluorouracil (5-FU), and

In a specific embodiment, the preferred order is to transfect the pUC19DNA or at least one of its valid substitutes first and, then,administering the chemotherapy drugs after the transfection of pUC19DNA.However, the pUC19 DNA transfection or at least one of its validsubstitutes and administering the chemotherapy drugs may be administeredto the cancer cells or patient concurrently or sequentially. In otherwords, the pUC19 DNA transfection may be administered first; thechemotherapy drugs may be administered first.

Cancers that may be treated using the present combinatorial protocolinclude, but are not limited to those carcinomas and sarcomas set forthherein above.

Combined Treatment of Apigenin and Stattic Synergistic Inhibition ofCal27 Cell Survival And Induced Cell Death.

In addition to NF-B, Signal transducer and activator of transcription(Stat) is another family of transcription factors. They mediate extracellular signals stimulated by cytokines and growth factors,translocation to the cell nucleus where they act as transcriptionactivators. These proteins mediate the expression of a variety of genesin response to cell stimuli, and thus play a key role in many cellularprocesses such as cell growth and apoptosis. Stat, such as STATS, playan important role in cancer cells survival and proliferation. However,Stat Inhibitors or IKK inhibitors alone showed little inhibiting effecton cancer cell survival. Evidence showed that these two transcriptionfactors interact with each other and to functionally cooperate with eachother. In addition, NF-κB and STAT binding sites linked together to formpromoter modules. Combination of Stattic, a Stat inhibitor with eitherIKK inhibitor or apigenin results in synergetic induction of cell death.This combination provides a method of treating cancer.

The present invention provides additional methods for inducing cancercell death and tumor suppression. In accordance with the presentinvention, it has been discovered that the combination of a IKKinhibitor or a CK2 inhibitor in combination with Stat inhibitor,stattic, or at least one of the valid substitutes for stattic actsynergistically to induce cancer cell death and to suppress tumorgrowth. Accordingly, the present invention provides a pharmaceuticalcomposition for the treatment of cancer in a subset of cancer cellsand/or in a patient comprising at least one IKK inhibitor or at leastone CK2 inhibitor and stattic or the at least one of the validsubstitutes for stattic in a pharmaceutically acceptable carrier. Alsoprovided is a method for treating cancer in a patient by administeringan effective amount of at least one IKK inhibitor or at least one CK2inhibitor in combination with stattic or valid substitutes. Suitable IKKinhibitors are as listed above. Suitable CK2 inhibitors, include, butare not limited to: Apigenin. Suitable Stat inhibitors are theinhibitors that inhibit stat phosphorylation, activation and nucleartranslocation, include, but not limited to stattic. The administrationof the IKK inhibitors or the CK2 inhibitors and the stattic may beadministered in any order. The preferred order is to administrate theinhibitors concurrently.

Advantages

From the description above, a number of advantages of the embodiments ofthis cancer treatment protocol and composition become evident:

This combination treatment targeting the tPMET and the HIF or cellresponses to hypoxia is a synergistic combination strategy that blockcancer cell respiration through the tPMET at cell surface while inhibitcancer cell capability of tolerating hypoxia. This combination did notinhibit cancer cell growth, but induced synergistic cancer specific celldeath. This combination composition and method represent a new conceptand principle for a new avenue of cancer treatment strategy for asynergistic cancer specific treatment and anti-cancer drug development.

The chemical structure of WST-3 represents a model of a class ofcompounds that is capable of interfering the tPMET and restricts itsactivity on cell surface without affecting the mitochondrial in thenormal cells. As cancer cells rely on cell surface ixygen consumption,the WST-3 represents the model of compounds that selectively affectcancer cells only.

The use of WST-1r also represents a novel strategy that incorporatecellular response to the treatemtn into the treatment protocol byinducing cancer cell tPMET followed by withdraw to induce cancer cellsdeath.

This combination treatment is different from conventional chemotherapythat inhibits cancer cell growth, instead, it directly induce cancercell death, which made it a more efficient cancer treatment byselectively killing cancer cells.

In summary this present invention provides a new concept ofcombinational treatment strategy for anticancer drug development. Thiscombination treatment will selectively block the cell surfacerespiration of cancer cell while inhibiting their capability to responseto hypoxia threofre, to inhibit cacner cell respiration and hence theenergy metabolism from two direction to obtain synergistic induciblecancer cell death. In addition, these treatments utilize non cytotoxiccompounds result in synergistic cancer specific cell death, whichprovides a new avenue for anti-cancer drug development and for cancertreatment.

Although the description above contains much specificity, these shouldnot be construed as limiting the scope of the embodiments but as merelyproviding illustrations of some of the presently preferred embodiments.For example, the WST-3 and the apigenin each represents classes ofchemical compounds with similar function. Also the combination of WST-3and apigenin represents a new strategy and a new avenue of cancer drugdevelopment by targeting tPMET in combination with inhibition ofcellular responses to hypoxia and some other related process.

Thus the scope of the embodiments should be determined by the appendedclaims and their legal equivalents, rather than by the examples given.

V. Administration of Pharmaceutical Compositions and Compounds

The pharmaceutical compositions can be administered by any suitableroute, for example, by injection, by intra vaneus infusion, by intraartery infusion, by oral, pulmonary, nasal, transdermal or other methodsof administration. In general, pharmaceutical compositions of thepresent specification comprise, among other things, pharmaceuticallyacceptable diluents, preservatives, solubilizers, emulsifiers, adjuvantsand/or carriers. Such compositions can include diluents of variousbuffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionicstrength; and additives such as detergents and solubilizing agents(e.g., Tween 80, Polysorbate 80), anti-oxidants (e.g., ascorbic acid,sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol)and bulking substances (e.g., lactose, mannitol). The compositions canbe incorporated into particulate preparations of polymeric compoundssuch as polylactic acid, polyglycolic acid, etc., or into liposomes.Such compositions may influence the physical state, stability, rate ofin vivo release, and rate of in vivo clearance of components of apharmaceutical compositions. See, e.g., Remington's PharmaceuticalSciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages1435-1712 which are herein incorporated by reference. The pharmaceuticalcompositions can be prepared, for example, in liquid form, or can be indried powder form (e.g., lyophilized). Particular methods ofadministering pharmaceutical compositions are described hereinabove.

In yet another embodiment, the pharmaceutical compositions can bedelivered in a controlled release system, such as using an intravenousinfusion, an implantable osmotic pump, a transdermal patch, liposomes,or other modes of administration. In a particular embodiment, a pump maybe used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. (1987)14:201; Buchwald et al., Surgery (1980) 88:507; Saudek et al., N. Engl.J. Med. (1989) 321:574). In another embodiment, polymeric materials maybe employed (see Medical Applications of Controlled Release, Langer andWise (eds.), CRC Press: Boca Raton, Fla. (1974); Controlled DrugBioavailability, Drug Product Design and Performance, Smolen and Ball(eds.), Wiley: New York (1984); Ranger and Peppas, J. Macromol. Sci.Rev. Macromol. Chem. (1983) 23:61; see also Levy et al., Science (1985)228:190; During et al., Ann Neurol. (1989) 25:351; Howard et al., J.Neurosurg. (1989) 71:105). In yet another embodiment, a controlledrelease system can be placed in proximity of the target tissues of theanimal, thus requiring only a fraction of the systemic dose (see, e.g.,Goodson, in Medical Applications of Controlled Release, supra, (1984)vol. 2, pp. 115-138). In particular, a controlled release device can beintroduced into an animal in proximity of the site of inappropriateimmune activation or a tumor. Other controlled release systems arediscussed in the review by Langer (Science (1990) 249:1527-1533).

The conclusion that this is programmed cell death is formed by theobservation that normal cells had no cytotoxic reaction and further,that a over 90% kill rate is more than substantial evidence of asignificant find. Therein, because this specification touches aprogrammed cancer cell death pathway that was prior untouched, or in thealternative, that this invention may activates a known pathway or anovel unknown pathway in a manner not able to be duplicated by otherinventions, the very sequence of events defined in this specificationactivates programmed cell death in the cancer cells and as such,presents a valid model for further study. In other words, throughprocesses known to those of skill, the very core molecular event leadingto the over 90% kill rate, can be explored because we have the workingmodel to induce such events. Therein, the invention is also claimed asan important model for further research, study and pathwayillumination/elucidation.

Although above and below I have shown specific experimentation and data,one of skill in the art of cancer preclinical and clinical protocolstructure, execution and analysis will recognize upon reading thisdocument, through variation of the dosages of the named components, theorder in which they are applied and the time frames betweenapplications, valid substitutions of the named components there are amyriad of variable applications which may result in the same or similaroutcome. To the extent that these variables can be applied to any cancerin any mammal, the inventor notes than nothing contained within thisdocument or any subsequent documentation provided by the inventor isintended to be limiting. The inventor also notes that this specificationis intended to work alone, and reduce cytotoxic effects of traditionalcancer therapy, such as chemotherapy and radiation, however, nothingherein is intended to limit the use of this specification to the extentthat chemotherapeutic and radiation combination therapies can beutilized in combination with this specification. Further, that the useof chemotherapy and radiation therapy combinations, in conjunction withthis specification, may reduce the cytotoxicity of the chemotherapy orradiation therapy because the dosages of the chemotherapy and radiationtherapy can be reduced when used in combination with this specification.And finally, that the named sprcification may further sensitize cancercells selectively over normal cells such that subsequent application ofchemotherapy and radiation, as well as combination chemo/radiationtherapies, will work more efficiently again, allowing for the reductionof chemotherapy and radiation and combination chemo/radiation dosages.

The foregoing description of the present specification providesillustration and description, but is not intended to be exhaustive or tolimit the specifications to the precise one disclosed. Modifications andvariations consistent with the above teachings may be acquired frompractice of the specification. Thus, it is noted that the scope of theinvention is defined by the claims and their equivalents.

The present specification will now be illustrated in more detail in thefollowing examples. It is to be understood that these examples serveonly to describe the specific embodiments of the present specification,but do not in any way intend to limit the scope of the claims. It is offurther note to one of skill that unique sequence data has been providedin this application. To the extent that each of these new sequence datarepresent novel targets for the development of cancer therapeutics,nothing contained herein is intended to be limiting. Said targets arenoted as potential targets for further development under thisapplication using the above methods and other methods known to those ofskill. Although not mentioned in this specification elsewhere, use ofradiation as a distinct step, or other small molecule drugs, DNA, RNA,siRNA and all other methods for cancer therapy known to those of skillare noted as possible adjuvant to these protocols.

VI. Examples Example 1 Synergistic Inhibition of NF-KAPPAB Activity

Overview: Normally, NF-kappaB activity is measured by reporter assay,electronic gel mobility shift assay and more recently, DNA bindingELISA. However, all of these methods employ exogenous DNA oligo orconstructs carrying consensus NF-kappaB response element sequences formeasuring specific NF-KAPPAB DNA binding and transcriptional activity.Additionally, the NF-KAPPAB consensus response element is different fromthe real promoter sequences that also need complex interaction withmultiple molecules and may introduce artificial effects.

Method: UM-SCC-6 cells were transfected with effectene (Qiagen) i) 20%dominant negative IKK1-KA (K44A) and 80% pUC19, ii) 20% dominantnegative IKK2-KA (K44A) and 80% pUC19, iii) 20% dominant negativeIKK1-KA (K44A), 20% dominant negative IKK2-KA (K44A) and 60% pUC19, iv)20% pcDNA3 and 80% pUC19 as negative control, for 72 hours. At the endof transfection, cells were lysed with lysis solution from GeneSpectrakit (Panomics). The I B, p100, CSNK2B mRNA levels were measured with theGeneSpectra kit. The expression levels of each transcript from differenttransfections were normalized by their 18sRNA level as measured at thesame time.

Previously, we observed partial inhibition of NF-KAPPAB reporteractivity by −50% caused by cotransfection of kinase dead K44A-IKK1 orK44A-IKK2 into UM-SCC-6 cells and other head and neck squamars carcinomacells. By measuring expression levels of endogenous NF-KAPPAB downstreamgene, I B, p100 and CK2, as an indicator of NF-KAPPAB activity, weobserved little inhibitory effect on NF-KAPPAB activity from K44A-IKK1transfected cells (˜20%) and no inhibitory effect from K44A-IKK2transfected cells. In contrast, when inhibiting both IKK1 and IKK2molecules by cotransfecting dominant negative K44A-IKK1 and K44A-IKK2simultaneously we observed ˜90% inhibition at all three target geneexpression levels that we measured (FIGS. 2 A and B). These data showedsynergistic inhibitory effect of combination of K44A-IKK1 and K44A-IKK2on constitutive NF-KAPPAB activity in these cancer cells, suggestingpotential interchangeable function between these two IKKs.

Example 2 Simultaneous Inhibition of IKK1 and IKK2 Also Lead to CancerCell Death

In addition to the inhibition of NF-KAPPAB activity, cell deathassociated with cotransfection of K44A-IKK1 and K44A-IKK into UM-SCC-6cells (FIG. 3). 48 hours after tranfection, K44A-IKK1 and K44A-IKK2co-transfected cells showed 85% reduction in cell number (FIG. 3 WST-1no) and dramatic cell death (FIG. 3B). The data represents the averageof 7 sets of duplicates. This result indicates that inhibition ofNF-KAPPAB activity does lead to cancer cell death and that this can bereached only by inhibiting both IKK1 and IKK2 simultaneously. Inaddition, adding tetrazolium dye WST-1r further enhanced cancer celldeath caused by double inhibition of IKKs (FIG. 3 WST-1-yes). Followinginhibition of both IKK1 and IKK2 treating cells with WST-1r furtherenhance cell death (FIG. 3 WST-1-yes). In FIG. 3A about 80% reduction ofcell number in K44A-IKK1 and K44A-IKK2 cotransfected cells and over 95%reduction when these cells were treated with WST-1 in addition tocotransfection of K44A-IKK1 and K44A-IKK. Data represents an average of7 sets of duplicates. FIG. 3B shows cell death from double transfectedcells, partial cell death from K44A-IKK1 or K44A-IKK2 single transfectedcells and further enhanced cell death by adding WST-1r treatment toK44A-IKK1 and K44A-IKK2 double trasfected cells.

Example 3 Wst-1 Promote HT1080 Human Sarcoma Cell Death by Combinationwith DNA Transfection and IKK Inhibitor Treatemnt

Methods: HT1080 cells were cultured in 96 well plates and transfectedwith one of the pUC19, pcDNA3, IKK1-KA, IKK1-KA+PUC19, or pcDNA3+pUC19DNA vectors for 24 hours followed with treatment of IKK Inhibitor IIIIat 3-30 μM for another 24 hours and, then, treated with 10% WST-1 for 4hours and cultured overnight before detection. The same treatments ofcells were measured at 24, 48 and 96 hours after WST-1 treatment. Cellviability was measured by Cell Count Kit 8 (CCK8).

Data showed (1) significant IKK inhibitor III dose dependent inductionof cell death from the cells that were transfected with any of the DNAvectors at 24, 48 and 96 hour after WST-1 treatment comparing to nontrasnfected control cells, but no significant difference between pUC19vector only from IKK1-KA vector (FIG. 4); (2) further induced cell deathand decreased cell survival detected from the cells that were treatedwith WST-1 at 24, 48 and 96 hours after WST-1 treatment comparing tothose with no WST-1 treated cells; (3) at 96 hours after the treatmentof WST-1 all the non transfected cells grow back to the same amount asthe untreated control, while partial recovery of the cells from no WST-1treated, but trasnfected cells; and (4) at 96 hours after all thetreatment, only the cells transfected and also treated with WST-1remained died with no recovery, which indicated the combination ofeither IKK inhibitor and/or DNA transfection with the WST-1 treatmentfurther synergize these cancer cells to 100% death. The difference inthe absorption at 24 hour after WST-1 treatment were caused, in partial,by decreased response from the WST-1 treated cells to the CCK8detection. This effect reduced in 48 hours after the removal of WST-1treatment and diminished at 96 hours after the removal of WST-1treatment. Morphology examination of the cells found that at 24 hoursafter the WST-1 treatment majority of the 30 μM IIKK inhibitor IIItreated cells died after the treatment. However, the survival cells thatwere not treated with WST-1 grow back up again. Conversely, the deathsof all the cells that were transfected with DNA vectors and with thesame IKK inhibitor III treatment and treated with WST-1 were 100%. Thesedata demonstrate the effect of WST-1 enhances the IKK inhibitor IIIinduce cancer cell death effect and promote cell death of these cellsand that pUC19 vector also contribute to the combined effect of inducingcell death.

Example 4 IFN Substitute pUC19 Transfection to Enhance IKK Inhibitor IIIand WST-1 Effect

Overview: Our previous data suggest that DNA transfection plays a rolein the triple combination treatment for synergistic cancer cell death.Moreover, Interferon (IFN) responses have been reported to be involvedin transfection effects. We examined whether IFN can be a substitute forthe DNA transfection effect for in vivo treatment.

Methods: HT1080 cells were cultured in 96 well plates and treated withIFN, IKK inhibitor III and WST-1 sequentially. Each set of the cellswere treated with one of the IFN members at the concentration rangingfrom 2-1000units/ml for 24 hours followed by IKK inhibitor III treatmentat concentration of 3-30 μM for another 24 hours and, then, with WST-1for 4 hours and cultured overnight before detection. Cell viability wasmeasured by CCK8 kit at 24 and 48 hours after WST-1 treatment. Total of15 IFNs were tested. They arewer IFN A, IFN B, IFN C, IFN D, IFN F, IFNG, IFN H, IFN I IFN J, IFN K, IFN 4b, and IFN WA, IFN, IFN and IL-6.

Representative data (FIG. 21) showed IFN dose dependent and IKKInhibitor III dependent decrease of cell growth and enhancement of celldeath comparing to that without IFN treatment. Comparing to pUC19 DNAtransfection, which synergized the inhibition of cell growth andpromotes cell death, IFN reached 80-90% inhibitory effects caused bypUC19 DNA transfection when combined with 30 μM BMS345541 and WST-1 at48 hours after WST-1 treatment.

Example 5 WST-1 Induces ROS Generation

Overview: WST-1 was first described by ishiyama et al in 1996 (IshiyamM, et al Biol Pharm Bull 1996, 19:1515-20). It is a cell proliferationdetection reagent manufactured by Roche. WST-1 is composed oftetrazolium salt WST-1{4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedilsulfonate} and an electron coupling reagent diluted in phosphate buffered saline.WST-1 can be cleaved by mitochondrial succinate-tetrazolium-reductasesystem. This cleavage has been used as the basis of the measurement oflive cell. However, WST-1 has been found impermeable to cell membraneand their reduction occurs at the cell surface or at the level of theplasma membrane via trans-plasma membrane electron transport (Berridge,M V et al, Biotechnol Annu Rev, 2005; 11:127052). Alternatively, WST-1can be reduced by cell surface NAD(P)H-Oxidase (Berridge, M V, et al,Antioxid Redox Signal, 2000, 2:231-42, Scalett, D J, et al, Biofactors,2004, 20:199-206) In the present invention WST-1 has been found tosynergize the inhibitory effect on cell growth and promote cancer celldeath when it is used in combination with at least one of the DNAtransfection, or IFN, or siRNA transfection and one of the IKK, orcombination of CK2 and GSK3 inhibitors. Theoretically, it has beenproposed that the balance between JNK activation and NF-KAPPAB activitydetermines cell faith to death or a live. Prolonged JNK activationinduces programmed cell death. Generation of ROS induces JNK activationwhile NF-KAPPAB activity leads to suppress ROS level (Luo, J L, et al,J. Clin. Invest, (2005) 115:2625-32, Shen, H M, et al, Free RadicalBiology & Medicine 2006, 40:928-939). The present invention has foundthat WST-1 induces ROS production in these cancer cells and promotescell death.

Methods: HT1080 cells were cultured in cover slices and transfected withpUC19 and treated with IKK inhibitor III in sequential. Following thesetreatment, the cells, thus, treated, were either labeled withCM-H2-DCFDA, a fluorescence dye that can labeling ROS in cells, and,then, treated with WST-1 for 30 minutes (FIG. 23A) or treated with WST-1for 2 hours and then labeled with CM-H2-DCFDA (FIG. 23-B). The resultswere recorded by a digital camera with Spotlight software. Manuelexposure levels were used to maintain the same exposure level forcomparison.

In both experiments, significant WST-1 induced ROS generation has beendocumented (FIGS. 23 A and B). In FIG. 23-A, we also observed IKKinhibitor III dose dependent labeled ROS from the cells that weretransfected with pUC19, and treated with IKK, but with no exposure toWST-1. This may suggest that IKK Inhibitor III may also induce ROSgeneration.

Example 6 LiCl+Apigenin Induced Synergized Cacner Cell Death, WST-1Enhance Further these Cell Death

Overview: LiCl is known to inhibit GSK3 activity and Apigenin is a multisignal transducer that inhibits multiple signaling processes, includingprotein kinase 2 (CK2). The activity of both GSK3 and CK2 are known toenhance constitutive NF-κB activity. This test was intended to examinewhether combination of LiCl and Apigenin can substitute DNA transfectionfor the synergistic inhibitory effect and induction of cancer celldeath.

Methods: UM-SCC-6 cells were cultured in 96 well plates and treated withLiCl (1, 3, 10, 30, 100 mM) and Apigenin (1, 3, 10, 30, and 100 μM) indifferent combination of their doses for 24 hours followed by WST-1treatment. Cell viability was measured with CCK8 kit.

Data showed that combination of LiCl and Apigenin dose dependentdecrease of cell growth and increased cell death comparing to untreatedcontrol cells. 10 μM AP and 100 mM LiCl showed synergistic increase ofcell death (FIG. 5A). The subsequent treatment of WST-1 further enhancedthis inhibitory effect (FIG. 5B).

Example 7 pUC19 DNA Transfection Synergize Chemotherapeutic Drug Effectin UM-SCC6 Cells

UM-SCC-6 cells were transfected with pUC19 DNA, pcDNA3, pUC19+pcDNA3,IKK1-KA+pUC19, IKK2-KA+pUC19, and IKK1-KA+IKK2-KA+pUC19, IKK1-KA+pcDNA3,IKK2-KA+pcDNA3, or IKK1-KA+IKK2-KA+pcDNA3, for 48 hours and, treatedwith variable doses of 5-FU (FIG. 6A) or Cis-Platinum (FIG. 6B) for 96or 72 hours respectively. Cell viabilities were measured in 72 and 96hours respectively after drug treatment. Data showed that pUC19transfected cells showed the strongest inhibitory effects on cellgrowth.

Example 8 pUC19 DNA Transfection Synergize Chemotherapeutic Drug Effectin HT1080 Cells

HT1080 Cells were transfected with pUC19 DNA, pcDNA3, pUC19+pcDNA3,IKK1-KA+pUC19, IKK2-KA+pUC19, and IKK1-KA+IKK2-KA+pUC19, IKK1-KA+pcDNA3,IKK2-KA+pcDNA3, or IKK1-KA+IKK2-KA+pcDNA3, for 48 hours before thetreatment of chemotherapy drugs at various doses. Cell viability wasmeasured in 72 and 96 hours after drug treatment.

Drug treatment: Cis-Platinum 30 ng/ml-3 μg/ml (FIG. 6D), Paclitaxel 1nM-10 μM (FIG. 6C), 5-FU 50 nM-500 μM (data not shown), Doxorubicin 30nM-3.3 μM (data not shown).

Variable enhancement and synergistic effects were shown by thetransfection of these DNA vectors. pUC19 DNA alone transfection showedthe strongest synergistic efficacy effect to this chemotherapy drugscomparing to other DNA vectors tested. IC50 of the drugs were loweredapproximately 10 fold when combining pUC19 DNA transfected cells to thatof untransfected cells of drug treatment (IC50 of Cis-Platinum fromHT1080 cells and UM-SCC-6 cells untransfecte control 3 μg/ml, pUC19transfected cells 0.3 μg/ml and 1 μg/ml respectively; IC50 of 5-FU fromuntransfected UM-SCC6 cells was more than 1 mM, but 200 μM from pUC19transfected cells; IC50 of texal from pUC19 transfected HT1080 cells was20 nM, while untransfected did not show any response upto 100 μM.).Furthermore, at 96 hours after of the cis-Platinum or palitaxeltreatment untransfected cells recovered and grown up while the celldeath from DNA transfected cells, especially pUC19 vector alonetransfected cells were irreversible, meaning they were 100% died. Thesedata suggest that these chemotherapy drugs inhibit cancer cell growth,but may not kill these cells. The combination of transfection of pUC19DNA promote cell to death.

Example 9 Combination Treatment of Apigenin with WST-1r SynergizesInduced Cancer Cell Death

Method: UM-SCC6, MDA-MB-231, Cal27, HT1080, T294, B6-5 and A431 cellswere treated with 3% or 10% of WST-1r or 10, 30, or 100 μM apigenin orcombination of variable concentrations of WST-1r with apigenin inparallel with untreated control cells and DMSO control for 4 hours,then, the treatments were removed and the cells, thus, treated werechanged to normal growth medium and maintained in culture for another 24hours. DMSO was used as vehicle control. Cell viabilities were measuredby CCK8 Kit and normalized to % of untreated control calls.

Result: DMSO treated cells of every tested cell line showed same levelsof cell viability as untreated control cells (data not shown).

A: UM-SCC6, MDA-MB-231, Cal27, HT1080, T294, B6-5 and A431 cells weretreated with 3% of WST-1r or 100 μM apigenin or combination of 3% WST-1rwith100 μM apigenin in parallel with untreated control cells. Dateshowed that the combination of WST-1r and apigenin induced 75% to 95%cell death of all seven tested cancer cell lines comparing to untreatedcontrols (FIG. 8A).

B-E: Data showed both WST-1r and apigenin dose dependent cell death andsynergized cell death effect when combining 3% WST-1r with 100 μMapigenin or 10% WST-1r with 30 μM apigenin B & C: WST-1r Dose-Responseof MA-MB-231 cells (B) and A431 cells (C). The IC50 of WST-1r in thepresence of 100 μM apigenin were 1% for both MDA-MB231 cells and A431cells, while treatment of apigein alone showed little effect on cellsurvival. D & E: apigenin Dose-Response of MDA-MB-231 cells and A431cells. The apigenin IC50 in the presence of 10% WST-1r were 10 μM forboth cell lines (FIG. 8B-E).

Example 10

Comparison of cell responses to modified WST-1r and Apigenin combinationtreatment between cancer cells and non-cancer cells. UM-SCC6, Cal27human head and neck cancer cell lines and primary cultured humanbronchia keratinocytes (HEKa) as labeled were treated with 10% modifiedWST-1r (mPMS 20nM, WST-1c 1 mM) with (WST-1r 10) or without (WST-1r 0)10% modified WST-1r in combination with 0 (Apigenin 0), 30 (Apigenin 30)or 100 (Apigenin 100) μM Apigenin for 4 hours, then, changed to apigeninin the corresponding concentrations for another 24 hours. Data showedthat each of the WST-1r and the Apigenin single agent treatment hadlittle effect on cell viabilities (FIG. 9). Combination of WST-1r with100 μM Apigenin resulted in synergistic cell death in both UM-SCC6 andCal27 cancer cell lines, but not in paired non-cancer primary culturedHEKa cells (FIG. 9). These data demonstrated the cancer cell specificityof this combination treatment.

Example 11 Time Course and Dose Response of WST-1r and Dose-Response ofApigenin Involved in the Combination Treatment of WST-1r with Apigenin

Methods: Cal27 (A), HT1080 (B) and UM-SCC6 (C) cells were treated withvariable concentration (1%, 3% and 10%) of WST-1r as indicated for 0.5,1, 2, and 4 hours in combination with varible doses of apigenin (3, 10,30 and 100 μM) as indicated and, then, treated with the sameconcentration of apigenin for another 24 hours. Cell viabilities weremeasured by CCK8 Kit and normalized as % of that of untreated controlcalls.

Results: Data showed WST-1r time and dose dependent and apigenin dosedependent cell death from all three tested cell lines (FIG. 10A-C).Synergetic induction of cell death (over 80%) occurred at 10% WST-1rtreatment for 0.5 hour in combination with 100 μM apigeinin treatedCal27 and UM-SCC6 cells (FIG. 10A, C) and at 3% WST-1r treatment for 1hour in combination with 100 μM apigeinin treated HT1080 cells (FIG.10B). By increasing WST-1r treatment time, 80-90% cell death can bereached at 30 μM apigenin in combination with 4 hour 10% WST-1rtreatment from HT1080 and UM-SCC6 cells (FIGS. 10B&C) and at 30 μMapigenin in combination with 1 hour 10% WST-1r treatment from Cal27cells (FIG. 10C). (Ap=apigenin; uM=μM)

Example 12 Effect of Combination Treatment with IKK Inhibitor and WST-1ron Melanoma Cell Lines

Mehtod: SK-Mel-5 and T294 human melanoma cells were treated with WST-1rat 1% and 3% final concentration respectively as indicated for 4 hours,then, removed WST-1r by changing to normal growth medium and added IKKinhibitor III for another 24 hours. After 24 hours treatment with 3nMand 10 μM IKK inhibitor III respectively as indicated, cells werechanged to grow in normal growth medium for 48 hours before measuringcell viability by CCK8 Kit.

Result: Both SK-mel-5 cells (FIG. 11A), and T294 cells (FIG. 11B) showedWST-1r and IKK inhibitor BMS345541 dose dependent increase of celldeaths. Combination of 3% WST-1r and 10 μM BMS345541 further synergizedthe induction of cell (FIGS. 11A&B).

Whereas, the non-cancer primary cultured human keratinocytes wereresistant to this combination treatment (FIG. 11C).

Example 13 Effects of Treatment Order of WST-1r and IKK Inhibitor III(BMS345541) on Inducing Human Melanoma Cell Death

Method: T294 cells were treated with 3% of WST-1r in combination with 3or 10 μM BMS345541 respectively in different order as indicated. Cellviabilities were measured by CCK8 Kit and normalized as % of that ofuntreated control calls. Control: Cells were either untreated or treatedwith BMS345541only at the indicated doses for 24 hours and, then,changed to normal growth medium for another 24 hours before measuringcell viability. B→W: Cells treated with BMS345541only at the indicateddoses for 24 hours and, then, added WST-1r at 3% final concentration for4 hours, then removing the treatment and changed to normal growth mediumfor another 24 hours before measuring cell viability. W+B: Cells treatedwith 3% WST-1r and BMS345541 at the indicated doses for 4 hours and,then, removing the treatment and changed to normal growth medium foranother 24 hours before measuring cell viability. W+B→B: Cells treatedwith 3% WST-1r and BMS345541 at the indicated doses for 4 hours and,then, removing the treatment and added BMS345541 at the indicated dosesin normal growth medium for another 24 hours before measuring cellviability. W→B: Cells treated with 3% WST-1r for 4 hours and, then,removing the treatment and added IKK BMS345541 at the indicated doses innormal growth medium for another 24 hours before measuring cellviability.

Result: Data showed that W→B and W+B→B treatment orders synergizedinduction of cell death (FIG. 12).

Example 14 WST-1r and Apigenin Combination Treatment Induced JNKPhosphrylation

Method: UM-SCC6 cells were treated with WST-1r and apigenin at theindicated doses for 4 hours, and then phosphorylated JNK and total JNKwere measured in parallel with FACE Kit (Qiogen). The resulting datawere normalized to total cell number measured by crystal violet stainingThe phosphorylated JNK from each measurement were normalized to theratio of phosphorylated JNK over total JNK values.

Result: Data showed WST-1r and apigenin dose dependent induction ofphosphorylation of JNK in UM-SCC6 cells (FIG. 13). Combination of WST-1rand apigenin further increased JNK phosphorylation. The most significantincrease of JNK phosphorylation from UM-SCC6 cells occurred at thetreatment of 100 μM apigenin in combination with 3% or 10% WST-1r. Thisresult supports the hypotheses that the combination of WST-1r withapigenin treatment induced JNK activation.

Example 15 Dose Response of ROS Generation after Combination Treatmentof WST-1r and Apigenin and IKK Inhibitor III

Method: UM-SCC6 cells were labeled with 10 μM CM-H2-DCFDA for 15 minutesand then treated with WST-1r or CCK8 at the indicated amounts incombination with variable doses of apigenin (A) or IKK Inhibitor III (B)for 4 hours. Fluorescence at Ex485/Em535 were measured for detecting ROSgeneration that labeled by the CM-H2-DCFDA.

Result: Data showed WST-1r dose dependent induction of ROS generation(FIGS. 24A and B). On the other hand, CCK8 induced low and very limitedlevel of ROS generation with no relation to the CCK8 treatment dose at 4hours after the treatment. Apigenin alone showed no effect on ROSgeneration. However, combination of apigenin with 1% and 3% WST-1r didshow apigenin dose dependent, limited, but, steady increase on ROSgeneration from thus treated cells when comparing to that of thecorresponding doses of WST-1r only treated cells. (FIG. 24-A)Conversely, combination of 10% of WST-1r with apigenin resulted indecrease of ROS levels (FIG. 24-B). In addition, when combined withCCK8, apigenin also increase the ROS generation (FIG. 24-A). This effectis apigenin dose dependent.

Similarly, IKK inhibitor III alone and combination of WST-1r with IKKinhibitor III (FIG. 24-B) showed similar effect as apigenin did, where 5μM IKK Inhibitor increased ROS levels while 10 μM IKK Inhibitor IIIdecreased it (FIG. 24-B). However, IKK Inhibitor III had no combinedeffect with CCK8 on ROS levels.

Example 16 Time Course of ROS Generation after Combination Treatment ofWST-1r and Apigenin and IKK Inhibitor III

Method: UM-SCC6 cells were labeled with 10 μM CM-H2-DCFDA for 15 minutesand then treated with WST-1r (B & D) or CCK8 (A & C) at the indicatedamounts in combination with variable doses of apigenin (C & D) or IKKinhibitor III (A & B) for the time period from 15 minute up to 4 hours.At each time points as indicated, fluorescence at Ex485/Em535 weremeasured for detecting ROS generation that labeled by the CM-H2-DCFDA.

Result: Data showed that WST-1r induced ROS generation continuedincrease and lasted at least for more than 4 hours (FIGS. 7B & D),whereas, CCK8 only induced low level and transience increase of ROS(FIG. 7-A & C).

Example 17 CCK8-XTT-WST-1 Comparison

Comparison cell death inducing capability of CCK8 and XTT to WST-1r incombination with apigenin treatment

Method: HT1080 and UM-SCC6 cells were treated with 10% of WST-1r, CCK8or XTT in combination with variable doses of apigenin for 4 hours and,then changed to normal growth medium for another 24 hours. Cellviability was measured with CCK8 kit.

Result: Data showed that CCK8 had no effect on cell death when comparingto control cells, while XTT showed intermediate induction of cell deatheffect comparing to WST-1r on both UM-SCC6 (FIG. 14B) and HT1080 cells(FIG. 14A). Apigenin IC50 of WST-1r and, XTT treated UM-SCC6 cells were5 and 25 μM while apigenin only and CCK8+apigenin treatments did notreached 1050. Similar result from HT1080 cells as well.

Example 18 Effects of Other Tetrazolium Salts as Substitutives of WST-1rfor Combination Treatment

Method: HT1080 and UM-SCC6 cells were treated with 1 mM WST-1, 0.4 mMWST-3, 0.5 mM WST-4, 0.5 mM WST-5 or 0.12 mM mPMS alone or each of theWST-3, WST-4, and WST-5 at the same concentration in combination with0.12 mM mPMS (0.4 mM WST-3+0.12 mM mPMS, 0.5 mM WST-4+0.12 mM mPMS, 0.5mM WST-5+0.12 mM mPMS) plus 10, 30 or 100 μM apigenin for 4 hours and,then changed to normal growth medium for another 24 hours. Cellviability was measured with CCK8 Kit.

Result: Data showed that WST-3 alone, WST-3+mPMS and WST-4+mPMS incombination with apigenin showed similar synergistic effect on inducingcell death that equivalent to that WST-1r does from both HT1080 cells(FIG. 17A) and UM-SCC6 cells (FIG. 17B). WST-1, WST-4, and WST-5 aloneshowed no such effect (FIG. 17 A,B). WST-3+mPMS are more potent thanWST-1r on cell death induction.

Example 19 mPMS Dose-Response

Method: A & B: HT1080 (FIG. 16A) and UM-SCC6 (FIG. 16B) cells weretreated with variable concentrations of mPMS as indicated in combinationwith 1 mM WST-1c and 10, 30 or 100 μM apigenin for 4 hours and, then,changed to normal growth medium for another 24 hours. Cell viabilitieswere measured by CCK8 Kit. 1 mM WST-1 only, 0.12 mM mPMS only and 10%WST-1r were used as parallel control. AP 0: Untreated Control, AP 10: 10μM Apigenin, AP 30: 30 μM Apigenin, AP 100: 100 μM Apigenin.

Result: Data showed mPMS and apigenin dose dependent cell death of bothHT1080 and UM-scc6 cells (FIGS. 16A &B). mPMS IC50 of combinationtreatment of apigenin 100 μM and mPMS+WST-1 from HT1080 cells was 5 μMverses 60 μM from untreated control cells. mPMS IC50 of combinationtreatment of apigenin 100 μM and mPMS+WST-1 from UM-SCC6 cells was 30 μMverses 80 μM from untreated control cells.

Example 20 Differential Cellular Responses to mPNS Treatment

Non cancer human keratinocyte (HEKa), SK-Mel-5 human malonoma cell line(SK5), human head and neck cancer cell Cal27 line (Cal27) and UM-SCC6line (SCC6) cells and human soft tissue sarcoma cell line HT1080(HT1080) were treated with 30, 40 or 50 μM mPMS for 4 hours and thencultured in normal growth medium for another 24 hours. Cell viabilitieswere measured with CCK8 kit. Data showed mPMS dose dependent cell deathand differential sensitivities to mPMS treatment from each of the celllines (FIG. 17). Among those, the non cancer primary cultured HEKa cellsshowed the least sensitivity to mPMS treatment with IC50 50 μM, whilethe IC50 from Cal27, UM-SCC6 and HT1080 cells were 20, and 30 μMrespectively.

Example 21 Effect of Combination Treatment of WST-3 with Apigenin onInduction of Cancer Cell Death

Method: UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treatedwith 50 or 100 μM WST-3 or 10 or 30 μM apigenin alone, or combination ofWST-3 and apigenin at different concentrations for 4 hours withuntreated cells as control, then, changed to normal growth medium andremained culture in this medium for another 24 hours. After the 24 hoursculture, cell viabilities were measured with CCK8 Kit. Data werenormalized to % of untreated control cells.

Result: A: Summary of differential cell responses to WST-3, apigenin andcombination treatments. Comparing to untreated cells (Ctrl) treatment of50 μM WST-3 (WST-3) or 30 μM apigenin (Apigenin) alone showed no orlimited effect of cell death to all tested cell line. Combination ofWST-3 and apigenin (Apigenin+WST-3) resulted in synergistic cell deathof SK-Mel-5, Cal27, UM-SCC6 and HT1080 all four tested human cancer celllines, but limited cell death from non cancer human keratnocytes (FIG.18A).

B & C: Comparison of Dose-Response of WST-3 and apigenin between noncancer HEKa and human melanoma cell line SK-Mel-5 cells. Data showedboth WST-3 and apigenin induced and dose dependent but limited celldeath from both HEKa cells (FIG. 18B) and SK-Mel-5 (FIG. 18C) cells.HEKa cells showed limited cell death in response to apigenin or WST-3alone treatment. WST-3 IC50 of combination of 30 μM apigenin and WST-3was 40 μM. Further increase WST-3 concentration showed no more celldeath from HEKa cells (FIG. 18B). However, the SK-Mel-5 cells showedsynergistic cell death response to combination treatment of 50 μM WST-3and 30 μM apigenin. The WST-3 IC50 from this combination treatment ofthe SK-Mel-5 cells was 20 μM, one fold less than that from HEKa cells(FIG. 18C). The HEKa cells were much more resistant to this combinationtreatment. Similar results were also observed from other cancer cells.

Example 22 Effect of Substitution of WST-1r with WST-3+mPMS forCombination Treatment with Apigenin on Induction of Cell Death

Method: UM-SCC6, HT1080, Cal27, SK-Mel-5, and HEKa cells were treatedwith 0.1 mM WST-3 plus 30 μM mPMS, or WST-3 only, or untreated controlin combination with 10 or 30 μM apigenin for 4 hours and, then, changedto normal growth medium and remained culture in this medium for another24 hours. After the 24 hours culture, cell viabilities were measuredwith CCK8 Kit. Data were normalized to % of untreated control cells.

Result: Data showed that over 90% induced cell death observed from thustreated Cal27, UM-SCC6, and SK-Mel-5 cells that were treated incombination of 0.1 mM WST-3, and 30 μM apigenin (FIG. 19C). Adding 30 μMmPMS to these treatments further synergize the cell death from Cal27 andUM-SCC6 cells (FIG. 19C). On the other hand, under this treatmentcondition, HEKa, primary cultured human keratinocytes, were relativeresistant to this treatment. This difference in sensitivity to thiscombination treatment may provide a window for differentiating targetingcancer cells and to control toxicity to normal cells.

Example 23 Enhancement of Taxel Efficacy Effects by Combination of Puc19DNA Sequence Derived siRNA with Taxel

Method: HT1080 cells transfected with siRNAs that were derived fromPuc19 DNA sequence and the siRNAs that targeting the corresponding genesthat are the targets of the Puc19 derived siRNAs for 24 hours, then,treated with Taxel at 3, 10, 30, and 100 μM for 48 hours. After the 48hours of Taxel treatment, cells in culture were changed to normal growthmedium for 24 to 72 hours. Cell viability was monitored by CCK8 Kitevery 24 hours. Data are normalized to % of untreated control cells. ThesiRNAs that used for this study includes siRNA#1, siRNA#2, siRHA#3,siRNA targeting TRPC6, SH3PXD2B, C6orf108, TTBK1, MAGI3, and TMEM182 aswell as combination of siRNA#2+#3, and siRNA#1+#2+#3+#4+#5 (siRNAΣ1-5).

Result: Data showed represent the measurements of 72 hours after thetreatment. The cell survival data showed Taxel dose dependent cell deathand enhanced cell death by Puc19 trasnfection and majority of the siRNAtrasfections (FIG. 22). The IC50 of taxel (Contrl IC50: 60 nM) wasreduced more than 3 fold by Puc19 DNA trasnfection (IC50: 20 nM) and bythe trasnfeciton of siRNAs targeting TRPC6 (IC50: 25 nM FIG. 22A),SH3PXD2B (IC50: 20 nM FIG. 22C), C6orf108 (IC50: 20 nM FIG. 22C), TTBK1(IC50: 35 nM FIG. 22C), MAGI3 (IC50: 20 nM FIG. 22A), and TMEM182 (IC50:20 nM FIG. 22B), as well as by the transfection of combination ofsiRNA#2+#3 (IC50: 25 nM FIG. 22A), B, and siRNA#1+#2+#3+#4+#5 (IC50: 20nM FIG. 22A-C). Over 2.5 fold IC50 decrease of taxel concentration wereobserved from siRNA#2 (IC50: 25 nM FIG. 22C), siRHA#3 (IC50: 25 nM FIG.22B), and siRNA targeting SH3PXD2B (IC50: 25 nM FIG. 22C), C6orf108(IC50: 20 nM FIG. 22C). These data demonstrated that the DAN sequencesof Puc19 DNA vector code for some short functional sequences that cantarget and interrupt the expression levels of the corresponding genesand the cellular functions. At least the genes (TRPC6, SH3PXD2B,C6orf108, MAGI3, and TMEM182) that have been tested can be used as atarget for anti-cancer drug design for enhancing the efficacy effect ofchemotherapy drugs. The siRNAs targeting these corresponding genes thatwere identified may also be used as a tool to reach this goal. Inaddition, these combined treatments induce cancer cell death rather thansimple inhibition of cell growth. 72 hours after treatment, the treatedcells did not grow back. This feature adds to lasting effect of thetreatment.

Example 24 Substitution of Puc19 with siRNA Against TMEM182 and MAGI3for Puc19-IKK Inhibitor-WST-1r Triple Combination Treatment

Method: HT1080 cells were transfected with siRNA#3, or the siRNAtargeting MAGI3, and TMEM182 that were derived from Puc19 DNA sequenceand the siRNAs that targeting the corresponding genes that are thetargets of the Puc19 derived siRNAs for 24 hours, then, treated with IKKinhibitor III for 24 hours followed by adding WST-1r for another 4 hour.After the 4 hours WST-1r treatment, cells in culture were changed tonormal growth medium for 24 hours. Cell viability were monitored by CCK8Kit every 24 hours. Data were normalized to % of untreated controlcells. AllStar siRNA was used as negative siRNA trasnfection control.Puc19 DNA vector transfciton was used as positive control.

Data label: Untreated control: 0 Ctrl, WST-1r only: 10 Ctrl, pUC19trasnfected cells: 0 p9, pUC19 transfected and WST-1r treated: 10 p9,AllStar negative contrl siRNA transfected: 0 AllStar, AllStartransfected and WST-1r treated: 10 AllStar, siRNA#3 transfected: 0siRNA#3, siRNA#3 transfcted and WST-1r treated: 10 siRNA#3, siRNAMAGI3transfected: 0 MAGI3, siRNA MAGI3 transfcted and WST-1r treated: 10MAGI3, siRNATMEM182 transfected: 0 TMEM182, siRNATMEM182 transfcted andWST-1r treated: 10 TMEM182,

Result: Data showed IKK inhibitor BMS345541 dose dependent cell deathand that siRNA targeting MAGI3 and TMEM182 synergize the cell death(FIG. 20). At 15 μM BMS345541 incombinatoin with 10% WST-1r and eitherpUC19 or the siRNA trasnfeection resulted in synergistic induction ofcell death. Again, these data showed that targeting TMEM182 and MAGI3may enhance effect on cancer treatment.

What is claimed:
 1. A pharmaceutical compound for treating cancercomprising: at least one functional group capable of inhibiting celltrans-plasma membrane electron transport (tPMET), inhibiting cellsurface respiration, uncoupling cell surface oxidative phosphorylation,or inhibiting tNOX activity; and at least one functional group capableof keeping the compound impermeable to cell plasma membrane, wherein thecompound does not affect mitochondria respiration in normal cells, theat least one functional group capable of uncoupling cell surfaceoxidative phosphorylation is selected from the group of oxidativeuncouplers consisting of: dinitrophenol (DNP),D5-chloro-3-tert-butyl-2′-chloro-4′-nitrosalicylanilide (S-13), sodium2,3,4,5,6-pentachlorophenolate (PCP),4,5,6,7-tetrachloro-2-(trifluoromethyl)-1H-benzimidazole (TTFB),Flufenamic acid (2-[3-(trifluoromethyl)anilino]benzoic acid),3,5-di-tert-butyl-4-hydroxy-benzylidenemalononitrile (SF6847), carbonylcyanide m-chloro phenyl hydrazone (CCCP), carbonyl cyanidep-[trifluoromethoxy]-phenyl-hydrazone (FCCP), andalpha-(phenylhydrazono)phenylacetonitrile derivatives, and weak acidscomprising comprising the chemical groups selected from the groupconsisting of weakly acidic phenols, benzimidazoles,N-phenylanthranilates, salicylanilides, phenylhydrazones, salicylicacids, acyldi-thiocarbazates, coumarins, aromatic amines, and cyanogroup.
 2. The pharmaceutical compound of claim 1, wherein the at leastone functional group capable of uncoupling cell surface oxidativephosphorylation comprises DNP.
 3. The pharmaceutical compound of claim1, wherein the at least one functional group capable of keeping thecompound impermeable to cell plasma membrane is selected from thechemical group consisting of the WST-1/WST-3, WST-4, WST-5, WST-8,WST-9, WST-10, WST-11, XTT, MTS,


4. The pharmaceutical compound of claim 1, wherein the at least onefunctional group capable of keeping the compound impermeable to cellplasma membrane comprises WST-3(2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disufophenyl)-2H-tetrazolium).5. A pharmaceutical composition for treating cancer comprising: a firstagent comprising a compound of claim 1; and a second agent that blocksat least one of HIF, cell hypoxia responses, NF-κB, C1(2 and IKKactivities.
 6. The pharmaceutical composition of claim 5, wherein thesecond agent comprises a flavonoid.
 7. The pharmaceutical composition ofclaim 5, wherein the second agent comprises apigenin.
 8. Thepharmaceutical composition of claim 5, wherein the second gent comprisesan IKK inhibitor, a CK2 inhibitor, a HIF inhibitor, or a tNOX inhibitor.9. The pharmaceutical composition of claim 13, wherein the IKK inhibitoris selected from the group consisting of BMS-345541, SC-514, IKK-2Inhibitor IV[5-(p-Fluoropheny0-2-uerido]thiophene-3-carboxamide, IKKInhibitor VII, IKK Inhibitor II, Wedelolactone, IKK-2 Inhibitor VN-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydrozybenzamide IMD-0354,and IKK-2 Inhibitor VI (5-Phenyl-2-ureido)thiophene-3-carboxamide. 10.The pharmaceutical composition of claim 5, wherein the second agentcomprises a pUC19, pcDNA3 (SEQ ID NO: 1 and 3) or siRNA selected fromthe group consisting of SEQ ID NOs: 10-12.
 11. A pharmaceuticalcomposition for treating cancer in a mammal in thereof comprising: afirst agent that blocks cell plasma membrane respiration, the firstagent being WST-1r(4-[3-(4-Iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzenedisulfonateand 1-methoxy-5-methyl-phanezinium methyl sulfate) or WST-3(2-(4-Iodophenyl)-3-(2,4-dinitrophenyl)-5-(2,4-disufophenyl)-2H-tetrazolium),a second agent that blocks at least one of NF-κB, CK2, IKK activitiesand cell hypoxia responses, and a pharmaceutically acceptable carrier.12. The pharmaceutical composition of claim 9, wherein the first agentis WST-1r or WST-3 and the second agent is selected from the groupconsisting of a flavonoid, apigenin, a NF-κB inhibitor, a HIF inhibitor,a CK2 inhibitor, an IKK inhibitor, a NOX inhibitor, a pUC19, PCDN3 (SEQID NO: 1 and 13), siRNAs selected from the group consisting of SEQ IDNOs: 10-12, and siRNAs targeting the expression of the genes selectedfrom the group consisting of SEQ ID NO: 2-5 and
 14. 13. Thepharmaceutical composition of claim 11, wherein the first agent isWST-1r and the second agent is apigenin.
 14. The pharmaceuticalcomposition of claim 11, wherein the first agent is WST-1r and thesecond agent is IKK inhibitor III.
 15. A method of selectively killingcancer cells comprising contacting a population of cells with acomposition of claim 5 in an amount effective to block the tPMET and/oroxidative phosphorylation and/or the coupling of the tPMET and theoxidative phosphryalation process and inhibiting cancer cell hypoxiaresponses.
 16. A method of selectively inhibiting cell surfacerespiration in cancer cells comprising contacting a population of cellswith a composition of claim 11 in an amount effective to block the tPMETand/or oxidative phosphorylation and/or the coupling of the tPMET andthe oxidative phosphorylation process.